Chimeric protein toxins for expression by therapeutic bacteria

BACKGROUND

Field of the Invention

This invention is generally in the field of therapeutic delivery systems including bacteria, and systems and methods for providing chimeric proteins efficiently targeted to cancer cells.

Description of the Prior Art

Citation or identification of any reference herein, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present application. The disclosures of each of these publications and patents are hereby incorporated by reference in their entirety in this application, and shall be treated as if the entirety thereof forms a part of this application.

Tumor-targeted bacteria offer tremendous potential advantages for the treatment of solid tumors, including the targeting from a distant inoculation site and the ability to express therapeutic agents directly within the tumor (Pawelek et al., 1997, Tumor-targeted Salmonella as a novel anticancer agent, Cancer Research 57: 4537-4544; Low et al., 1999, Lipid A mutant salmonella with suppressed virulence and TNF-alpha induction retain tumor-targeting in vivo, Nature Biotechnol. 17: 37-41). However, the primary shortcoming of tumor-targeted bacteria investigated in the human clinical trials (Salmonella strain VNP20009 and its derivative TAPET-CD; Toso et al., 2002, Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma, J. Clin, Oncol. 20: 142-152; Meir et al., 2001, Phase 1 trial of a live, attenuated Salmonella typhimurium (VNP20009) administered by direct Intra-tumoral (IT) injection, Proc Am Soc Clin Oncol 20: abstr 1043); Nemunaitis et al., 2003, Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients, Cancer Gene Therapy 10: 737-744) is that no significant antitumor activity has been observed, even in patients where the bacteria was documented to target the tumor. One method of increasing the ability of the bacteria to kill tumor cells is to engineer the bacteria to express conventional bacterial toxins (e.g., WO 2009/126189, WO 03/014380, WO/2005/018332, WO/2008/073148, US 2003/0059400 U.S. Pat. Nos. 7,452,531, 7,354,592, 6,962,696, 6,923,972, 6,863,894, 6,685,935, 6,475,482, 6,447,784, 6,190,657 and 6,080,849, 8,241,623, 8,524,220 8,771,669, 8,524,220, each of which is expressly incorporated herein by reference).

Use of protein toxins for treatment of various disorders including inflammation, autoimmunity, neurological disorders and cancer has long-suffered from off-target toxicity. Enhancing toxin specificity, which offers the potential to eliminate side effect, has been achieved by several different means, such as attachment of a specific antibodies or peptide ligand (e.g., Pseudomonas exotoxin A (PE-ToxA) antibody conjugate, known as an immunotoxin), or a ligand targeted to a surface molecule of the target cell. Based upon the binding specificity of the attached antibody or ligand moiety for a specific target, enhanced specificity of the target is achieved.

Other toxins have been engineered to achieve specificity based upon their sight of activation. For example, proaerolysin requires proteolytic activation to become the cytotoxic protein aerolysin. Substitution of the natural protease cleavage site for a tumor-specific protease cleavage site (e.g., that of the prostate specific antigen (PSA) protease or urokinase) results in a toxin selectively activated within tumors (Denmeade et al. WO 03/018611 and Denmeade et al. U.S. Pat. No. 7,635,682), specifically incorporated by reference herein. Another similar activation system has utilized ubiquitin fusion, coupled with a hydrolysable tumor protease (e.g., PSA) sequence and a toxin (e.g., saporin), as described by Tschrniuk et al. 2005 (Construction of tumor-specific toxins using ubiquitin fusion technique, Molecular Therapy 11: 196-204), also specifically incorporated by reference herein. However, while some specificity is engendered and thus these activated protein types are useful in the present technology as modified herein, in these types of engineered toxins, off-target toxicity can occur. In the case of the Pseudomonas immunotoxin, several dose-limiting toxicities have been identified. Vascular leakage syndrome (VLS) is associated with hypoalbuminemia, edema, weight gain, hypotension and occasional dyspnea, which is suggested to occur by immunotoxin-mediated endothelial cell injury (Baluna et al., 2000, Exp. Cell Res. 258: 417-424), resulting in a dose-limiting toxicity. Renal injury has occurred in some patients treated with immunotoxins, which may be due to micro-aggregates of the immunotoxin (Frankel et al., 2001, Blood 98: 722a). Liver damage from immunotoxins is a frequent occurrence that is believed to be multifactorial (Frankel, 2002, Clinical Cancer Research 8: 942-944). To date, antibodies linked to proteinaceous toxins have limited success clinically.

Recently developed approaches to delivery of therapeutic molecules (U.S. Pat. Nos. 8,241,623; 8,524,220; 8,771,669; and 8,524,220) have coupled a protease sensitive therapeutic molecule with co-expression of protease inhibitors, expressly incorporated by reference herein.

Use of secreted proteins in live bacterial vectors has been demonstrated by several authors. Holland et al. (U.S. Pat. No. 5,143,830) have illustrated the use of fusions with the C-terminal portion of the hemolysin A (hlyA) gene, a member of the type I secretion system. When co-expressed in the presence of the hemolysin protein secretion channel (hlyBD) and a functional TolC, heterologous fusions are readily secreted from the bacteria. The type I secretion system that has been utilized most widely, and although it is currently considered the best system available, is thought to have limitations for delivery by attenuated bacteria (Hahn and Specht, 2003, FEMS Immunology and Medical Microbiology, 37: 87-98). Those limitations include the amount of protein secreted and the ability of the protein fused to it to interfere with secretion. Improvements of the type I secretion system have been demonstrated by Sugamata and Shiba (2005 Applied and Environmental Micobiology 71: 656-662) using a modified hlyB, and by Gupta and Lee (2008 Biotechnology and Bioengineering, 101: 967-974) by addition of rare codons to the hlyA gene, each of which is expressly incorporated by reference in their entirety herein. Fusion to the gene ClyA (Galen et al., 2004, Infection and Immunity, 72: 7096-7106 and Type III secretion proteins have also been used. Surface display has been used to export proteins outside of the bacteria. For example, fusion of the Lpp protein amino acids 1-9 with the transmembrane region B3-B7 of OmpA has been used for surface display (Samuelson et al., 2002, Display of proteins on bacteria, J. Biotechnology 96: 129-154, expressly incorporated by reference in its entirety herein). The autotransporter surface display has been described by Berthet et al., WO/2002/070645, expressly incorporated by reference herein. Other heterologous protein secretion systems utilizing the autotransporter family can be modulated to result in either surface display or complete release into the medium (see Henderson et al., 2004, Type V secretion pathway: the autotransporter story, Microbiology and Molecular Biology Reviews 68: 692-744; Jose, 2006 Applied Microbiol. Biotechnol. 69: 607-614; Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333:1218-1226 and Rutherford and Mourez 2006 Microbial Cell Factories 5: 22). For example, Veiga et al. (2003 Journal of Bacteriology 185: 5585-5590 and Klauser et al., 1990 EMBO Journal 9: 1991-1999) demonstrated hybrid proteins containing the .beta.-autotransporter domain of the immunoglobulin A (IgA) protease of Nisseria gonorrhea. Fusions to flagellar proteins have been demonstrated. The peptide, usually of 15 to 36 amino acids in length, is inserted into the central, hypervariable region of the FliC gene such as that from Salmonella muenchen (Verma et al. 1995 Vaccine 13: 235-24; Wu et al., 1989 Proc. Natl. Acad. Sci. USA 86: 4726-4730; Cuadro et al., 2004 Infect. Immun. 72: 2810-2816; Newton et al., 1995, Res. Microbiol. 146: 203-216, expressly incorporated by reference in their entirety herein). Multihybrid FliC insertions of up to 302 amino acids have also been prepared (Tanskanen et al. 2000, Appl. Env. Microbiol. 66: 4152-4156, expressly incorporated by reference in its entirety herein). Trimerization of antigens can be achieved using the T4 fibritin foldon trimerization sequence (Wei et al. 2008 J. Virology 82: 6200-6208) and VASP tetramerization domains (Kuhnel et al., 2004 PNAS 101: 17027-17032), expressly incorporated by reference in their entirety herein. The multimerization domains are used to create, bi-specific, tri-specific, and quatra-specific targeting agents, whereby each individual agent is expressed with a multimerization tag, each of which may have the same or separate targeting peptide, such that following expression, surface display, secretion and/or release, they form multimers with multiple targeting domains.

SUMMARY OF THE INVENTION

Modified Therapeutic Molecules

The present technology, according to various embodiments, consists of known and/or novel chimeric proteins, or combinations of proteins, that are expressed, secreted, surface displayed and/or released by bacteria and result in anticancer activity or have direct inhibitory or cytotoxic anti-neoplastic activity, including activity against cancer stem cells and/or cancer mesenchymal stromal cells, and may optionally include the combination with secreted protease inhibitors. The bacterial delivery vector may be attenuated, non-pathogenic, low pathogenic (including wild type), or a probiotic bacterium. The bacteria are introduced either systemically (e.g., parentral, intravenous (IV), intramuscular (IM), intralymphatic (IL), intradermal (ID), subcutaneously (sub-q), local-regionally (e.g., intralesionally, intratumorally (IT), intrapaeritoneally (IP), topically, intrathecally (intrathecal), by inhaler or nasal spray) or to the mucosal system through oral, nasal, pulmonary intravessically, enema or suppository administration where they are able to undergo limited replication, express, surface display, secrete and/or release the anti-cancer inhibitory proteins or a combination thereof, and thereby provide a therapeutic benefit by reducing or eliminating the disease, malignancy and/or neoplasia.

The present technology, according to various embodiments, further consists of modified forms of toxins with improved secretion, surface display and/or release by the bacteria, and/or modifications that improve the overall activity and/or specificity of the toxin. Such toxins may be further co-expressed with protease inhibitors as previously described (See, U.S. Pat. Nos. 8,241,623; 8,524,220; 8,771,669; 8,524,220).

Toxins, therapeutic cytokines and other molecules, homologues or fragments thereof useful in conjunction with the present technology, according to various embodiments, includes small lytic peptides, larger lytic peptides, pore-forming toxins, protein inhibitors, extracellular DNAases (DNase), intracellular DNAases, apoptosis inducing peptides, cytokines, prodrug converting enzymes, metabolite destroying enzymes, ribonucleases, antibody inactivating toxins and other anticancer peptides. In a preferred embodiment, the toxins include those that are naturally secreted, released and/or surface displayed, or heterologously secreted, released and/or surface displayed, and that can be modified uniquely to suit the delivery by a bacterium and may be further engineered to have the tumor, lymphoma, leukemic bone marrow or proximity-selective targeting system described herein, including but not limited to the proteins azurin, carboxyesterase Est55 (a prodrug-converting enzyme from Geobacillus that activates CPT-11 to SN-38), thiaminase (e.g., from Bacillus), methionase (methioninase), asparaginase, tryptophanase, apoptin, Torquetnovirus (TTV) derived apoptosis-inducing protein TAIP and with gyrovirus VP3 bax, bim, p53, BAK, BH3 peptide (BCL2 homology domain 3), cytochrome C, thrombospondin, platlet factor 4 (PF4) peptide, Bacillus sp. cytolysins, Bacillus sp. nheABC toxins, cytolethal distending toxins (cldt) including those cldts from Haemophilus, Aggregatibacter, Salmonella, Escherichia, Shigella, Campylobacter, Helicobacter, Hahella and Yersinia, typhoid toxins (pertussis like toxin) (pltAB), pertussis toxin, cldt:plt hybrids, actAB, cytotoxic nectrotic factor (cnf), dermonecrotic factor (dnf), shiga toxins and shiga-like toxins, bacteriocins, (colicins and microcins; Hen and Jack, Chapter 13 Microcins, in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Nes et al., Chapter 17, The nonlantibiotic heat-stable bacteriocins in gram-positive bacteria, in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Sharma et al., Chapter 18 in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press) including membrane depolarizing (or pore-forming), DNAases (including colicin DNase, Staphylococcal Nuclease A:OmpA fusions (Takahara et al., 1985 J. Biol. Chem 260: 2670-2674), Serratia marcescens DNase (Clegg and Allen, 1985, FEMS Microbiology Letters 27: 257-262; Vibrio DNase Newland et al., 1985 Infect Immun 47: 691-696) or other bacterial DNase), RNAases, and tRNAases, including but not limited colicin A, colicin D, colicin E5, colicin E492, microcin M24, colE1, colE2, colE3, colE5 colE7, coleE8, colE9, col-Ia, colicin N and colicin B, membrane lytic peptides from Staphalococcus (listed below) and sea anemones, P15 peptide and other TGF-beta mimics, repeat in toxin (RTX) family members (together with the necessary acylation and secretion genes) including Actinobacillus leucotoxins, a leuckotoxin: E. coli HlyA hybrid, E. coli HlyA hemolysin, Bordetella adenylate cyclase toxin, heat stable enterotoxins from E. coli and Vibrio sp. (Dubreuil 2006, Chapter 48, Eschericia coli, Vibrio and Yersinia species heat stable enterotoxins, Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press), autotransporter toxins including but not limited to IgA protease, picU espC, and sat, Staphalococcus protein A, chlostridium enterotoxin, Clostridium difficile toxin A, scorpion chlorotoxin, aerolysin, subtilase, cereolysin, Staphalococcus leukotoxins (e.g. LukF-PV, LukF-R, LukF-I, LukM, HlgB) and the other, to class S (e.g. LukS-PV, LukS-R, LukS-I, HlgA, HlgC). Best known are the toxins produced by S. aureus: .gamma.-haemolysins, HlgA/HlgB and HlgC/HlgB and leukocidin Panton-Valentine, LukS-PV/LukF-PV (Luk-PV, PVL)) TRAIL, fasL, IL-18, CCL-21, human cyokine LIGHT, agglutinins (Maackia amurensis, wheat germ, Datura stramonium, Lycopersicon (tomato) plant lectin, leukoagglutinin (L-PHA, Helix pomatia) saporin, ricin, pertussus toxin, and porB, as well as other toxins and peptides (Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press; each of which is expressly incorporated by reference in their entirety herein). Metabolite toxins such as the Chromobacterium violacium dipsepeptides (Shigeatsu et al., 1994, FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. II. Structure determination. J Antibiot (Tokyo) 47(3):311-4) or those from Serratia are also of use in the present technology.

The chimeras may be further modified by addition of one or more multimerization domains, such as the T4 foldon trimerization domain (Meier et al., 2004, Journal of Molecular Biology, 344: 1051-1069; Bhardwaj et al., Protein Sci. 2008 17: 1475-1485) or tetramerization domains such as VASP (Kuhnel et al., 2004 PNAS 101: 17027-17032). Chimeric toxins may be further modified by the addition of known cell penetrating (ferry) peptide which further improves their entry into target cells. Cell penetrating peptides include those derived from the human immunodifficency virus (HIV) TAT protein amino acids 47-57 (YGRKKRRQRRR SEQ ID NO.: 020) and used in fusion proteins (e.g., TAT-apoptin, TAT-bim, TAT-p53), the antennapedia homeodomain (penetraxin), Kaposi fibroblast growth factor (FGF) membrane-translocating sequence (MTS), herpes simplex virus VP22, hexahistidine, hexylysine, hexaarginine or "Chariot" (Active Motif, Carlsbad, Calif.; U.S. Pat. No. 6,841,535). Nuclear localization signals (NLSs) may also be added, including but not limited to that from herpes simplex virus thymidine kinase, the SV40 large T antigen monopartite NLS, or the nucleoplamin bipartite NLS or more preferably, the NLS from apoptin, a tumor associated (tumor-selective) NLS. The tumor-selective nuclear export signal from apoptin may be used alone or together with NLS from apoptin (Heckl et al., 2008, Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells, Apoptosis 13: 495-508; Backendor et al., 2008, Apoptin: Therapeutic potential of an early sensor of carcinogenic transformation, Ann Rev Pharmacol Toxicol 48: 143-69).

Expression of wild type Pseudomonas exotoxin A has been previously described (U.S. Pat. Nos. 6,962,696; 7,452,531; 8,241,623; Swofford C A et al. 2014. Identification of Staphylococcus aureus .alpha.-hemolysin as a protein drug that is secreted by anticancer bacteria and rapidly kills cancer cells. Biotechnol Bioeng. 111:1233-45). Regarding chimeric Pseudomonas exotoxin A (ToxA), numerous forms have been generated and shown to be effective at killing tumor cells in vitro and in murine cancer models (TGF-.alpha.-GIII-KDEL (Kihara and I. Pastan 1994 Small Chimeric Toxins Containing Only Transforming Growth Factor .alpha. and Domain III of Pseudomonas Exotoxin with Good Antitumor Activity in Mice. Cancer Res 1994:54:5154-5159; TGF .alpha.-PE38; Kreitman et al., 1992, Properties of Chimeric Toxins with Two Recognition Domains: Interleukin 6 and Transforming Growth Factor .alpha. at Different Locations in Pseudomonas Exotoxin. Bioconjugate Chem. 1992, 3, 63-68; Kreitman et al., Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin 2 receptor-expressing human carcinoma. Blood 1994, 83, 426-434; Zielenski 2009, Affitoxin--A Novel Recombinant, HER2-Specific, Anti-Cancer Agent for Targeted Therapy of HER2-Positive Tumors. J Immunother. 32: 817-825). However, these investigators did not use a live bacterial vector to deliver the chimeric agent, and purified the agent from bacteria that were killed and lysed, generally requiring the biochemical manipulation of "refolding" of an inactively produced agent in the form of an inclusion body to produce the active form. The process of refolding an inactive inclusion body is not facilitated by live bacteria, and thus cannot be effective when delivered by a live bacterium expressing such a cancer therapeutic protein. Proteins produced in a "soluble" form, while not requiring refolding, likewise are ineffective because they do not get out of the cell. The present technology, according to various embodiments, uses, alone or in combination, ToxA domain Ib containing at least 2 available cysteines for cysteine bonds, flexible linkers, colicin lysis protein and/or signal sequences that result in functional ToxA chimeric proteins that are expressed, secreted, surface displayed and/or released by the bacteria and have the ability to kill cancer cells in the absence of biochemical manipulation.

Regarding use of tumor-targeted bacteria expressing wild type cytolethal distending toxin and chimeras including those with apoptin, there have been several earlier descriptions (U.S. Pat. Nos. 6,962,696; 7,452,531; 8,241,623; 8,524,220; 8,623,350; 8,771,669). Cytolethal distending toxins (CLDTs) comprise a family of heterotrimeric holotoxins produced by bacteria that are internalized into mammalian cells and translocated into the nucleus. CLDTs are known to occur in a number of bacterial genera including Haemophilus, Aggregatibacter, Salmonella, Escherichia, Shigella, Campylobacter, Helicobacter, Hahella and Yersinia (Gargi et al., 2012 Bacterial toxin modulation of the eukaryotic cell cycle: are all cytolethal distending toxins created equally? Frontiers in Cellular and Infection Microbiol. 2:124. doi: 10.3389/fcimb.2012.00124), however CLDT does not exist in the VNP20009 strain of Salmonella used in human clinical studies (Toso et al. 2002. Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma. J. Clin. Oncol. 20, 142-152; Low et al., 2004, Construction of VNP20009, a novel, genetically stable antibiotic sensitive strain of tumor-targeting Salmonella for parentral administration in humans. Methods Mol Med 90: 47-60).

Depending upon both the specific CLDT and the mammalian cells type, different effects have been documented. All CLDTs have homology to exonuclease III and several have been directly shown to exhibit DNase activity in vitro (Ewell and Dreyfus 2000 DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol Microbiol 37, 952-963; Lara-Tejero and Galan, 2000 A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Science 290, 354-357), which is believed to be the primary effect of the toxin. The DNase activity results in double-stranded DNA breaks that activates the cell's DNA damage response and interrupts the cell cycle at G2M. Non-haematopoetic cells tend to enlarge, hence part of the toxin name distending, and in many cases the cells subsequently undergo apoptosis. In haematopoitic cells apoptosis is more rapidly produced (Jinadasa et al., 2011, Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages. Microbiology 157: 1851-1875; Gargi et al., 2012).

Most of the CLDTs are organized in a unidirectional operon of cldtA, cldtB and cldtC genes, where the cldtB encodes the active subcomponent, and cldtA and cldtC encode peptides that are involved in cell binding and translocation. In Salmonella however, the genes exist as a bidirectional operon consisting of cldtB together with a two pertussis like toxin subunits oriented in the opposite direction, pltA and pltB, as well as sty and ttsA, also in opposing directions, that are reported to be required for secretion of the toxin (Hodak and Galan 2013 A Salmonella Typhi homologue of bacteriophage muramidase controls typhoid toxin secretion. EMBO Reports 14: 95-102). However, in the present technology, according to various embodiments, the presence of sty and ttsA are not required for secretion of the active toxin when the operon is reorganized into a unidirectional operon of cldtB, pltB and pltA.

Translocation of E. coli CLDTs to the nucleus, which constitutes the target location for the endonuclease activity, requires the presence of a nuclear localization signal (NLS). In Escherichia coli CLDT-II for example, the NLS is bipartite and located at the C-terminus (McSweeney and Dreyfus, 2004). Nishikubo et al., 2003 identified an NLS occurring in the 48-124 amino acid region in Actinobacillus actinomycetemcomitans.

Apoptins are a family of viral genes that were first discovered in chicken anemia virus. Apoptin is the product of the VP3 gene that is involved in lymphoidal atropy and anemia in infected chickens (Penaloza et al., 2014 Apoptins: selective anticancer agents, Trends in Molecular Medicine 20: 519-528; Los et al., 2009 Apoptin, a tumor selective killer, Biochimica et Biophysica Acta 1793: 1335-1342). Apoptin was subsequently found to selectively induce apoptosis in cancer cells (Danen-Van Oorschot et al., 1997 Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc. Natl. Acad. Sci. USA 94: 5843-5847). Apoptin shares similarity with Torquetnovirus (TTV) derived apoptosis-inducing protein TAIP and with gyrovirus VP3. Apoptin consists of several different domains including a leucine rich sequence (LRS) which is involved in binding to the promyelocytic leukemia (PML) protein of nuclei and in apoptin multimerization, an SRC homology 3 (SH3) binding domain with is part of a bipartite nuclear localization signal (NLS), a nuclear export sequence (NES) that promotes egress of apoptin from normal cell nuclei, a set of threonines of which T108 must be phosphorylated for full apoptin activity, the C-terminal portion of the bipartite NLS and an anaphase promoting complex/cyclosome 1 (APC/Cl) binding domain that consists of approximately one third of the C-terminus.

The present technology, according to various embodiments, consists of a modified Salmonella CLDT operon and forms of cytolethal distending toxins that are chimeric with apoptin and other peptide moieties including peptide linkers that improve activity and peptide blocking moieties that must be specifically cleaved to activate the protein. The present technology, according to various embodiments, uses deletions in the the CLDT nuclear localization signals which are then complemented by N- or C-terminal fusions with apoptin, or apoptin fragments, which supply its nuclear localization signal in trans as a fusion peptide.

The types of cancers or neoplasias to which the present technology is directed include all neoplastic malignancies, including solid tumors such as those of colon, lung, breast, prostate, sarcomas, carcinomas, head and neck tumors, melanoma, as well as hematological, non-solid or diffuse cancers such as leukemia and lymphomas, myelodysplastic cells, plasma cell myeloma, plasmacytomas, and multiple myelomas. Specific types of cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma adrenocortical carcinoma, adult (primary) liver cancer, adult acute myeloid leukemia, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, astrocytomas (childhood), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer (female), breast cancer (male), bronchial tumors, Burkitt's lymphoma, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, central nervous system tumors, cervical cancer, childhood acute myeloma, childhood multiple myeloma/plasma cell neoplasm, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, endometrial cancer, endometrial uterine sarcoma, ependymoblastoma, ependymoma, esophageal cancer, Ewing sarcoma family of tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic gallbladder cancer, eye cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (gist), germ cell tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular (eye) melanoma, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, Langerhans cell, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, lip and oropharyngeal cancer, liver cancer (metastatic), lung cancer (primary), macroglobulinemia, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, metastatic stomach (gastric) cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloproliferative disorders, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system atypical teratoid/rhabdoid tumor, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian gestational trophoblastic tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary cervical cancer, primary hepatocellular (liver) cancer, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (Basal cell carcinoma), Sezary syndrome, skin cancer (nonmelanoma), skin carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, teratoid/rhabdoid tumor (childhood), testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site, ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Waldenstrom malignant fibrous histiocytoma of bone and osteosarcoma, and Wilms tumor.

The therapeutic agent can be a chimera consisting of a peptide or protein, toxin, chimeric toxin, cytokine, antibody, bispecific antibody including single chain antibodies, camel antibodies and nanobodies chemokine, prodrug converting enzyme or metabolite-degrading enzyme such as thiaminase, methionase (methioninase, L-methionine .gamma.-lyase) or asperaginase. In a preferred embodiment the therapeutic agent is a toxin, or modified toxin.

The chimeras may be further modified by addition of one or more multimerization domains, such as the T4 foldon trimerization domain (Meier et al., 2004, Journal of Molecular Biology, 344: 1051-1069; Bhardwaj et al., Protein Sci. 2008 17: 1475-1485) or tetramerization domains such as VASP (Kuhnel et al., 2004 PNAS 101: 17027-17032). Chimeric toxins may be further modified by the addition of known cell penetrating (ferry) peptide which further improves their entry into target cells. Cell penetrating peptides include those derived from the HIV TAT protein (e.g., TAT-apoptin, TAT-bim, TAT-p53), the antennapedia homeodomain (penetraxin), Kaposi fibroblast growth factor (FGF) membrane-translocating sequence (MTS), herpes simplex virus VP22, hexahistidine, hexalysine, hexaarginine or "Chariot" (Active Motif, Carlsbad, Calif.; U.S. Pat. No. 6,841,535). Nuclear localization signals (NLSs) may also be added, including but not limited to that from herpes simplex virus thymidine kinase, the SV40 large T antigen monopartite NLS, or the nucleoplasmin bipartite NLS or more preferably, the NLS from apoptin, a tumor associated (tumor-selective) NLS. The tumor-selective nuclear export signal from apoptin may be used alone or together with NLS from apoptin (Heckl et al., 2008, Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells, Apoptosis 13: 495-508; Backendor et al., 2008, Apoptin: Therapeutic potential of an early sensor of carcinogenic transformation, Ann Rev Pharmacol Toxicol 48: 143-69).

The chimeric proteins may have one or more additional features or protein domains known to those skilled in the art which are designed to be active or catalytic domains that result in the death of the cell, allow or facilitate them being secreted or released by autolytic peptides such as those associated with colicins or bacteriophage release peptides have targeting peptides that direct them to the target cells, and protease cleavage sites for activation (e.g., release from parent peptide), and thioredoxin or glutathione S-transferase (GST) fusions that improve solubility.

The present technology also provides in accordance with some embodiments, unique chimeric modifications of the above listed toxins that contain specific combinations of components resulting in secretion by selective anti-tumor activity. The technology also provides extracellular protease sensitivity (deactivation) that may include the addition of protease cleavage sites and may be co-expressed with a protease inhibitor. The chimeric proteins may have one or more additional features or protein domains known to those skilled in the art which are designed to 1) be active or catalytic domains that result in the death of the cell or make them susceptible to other known anticancer agents, 2) allow or facilitate them being secreted or released by autolytic peptides such as colicin release peptides, 3) membrane protein transduction (ferry) peptides, 4) autotransporter domains, 5) have targeting peptides that direct them to the target cells, and 6) protease cleavage sites for activation (e.g., release from parent peptide). However, the specific organization and combination of these domains is unique and specific to the technology.

Small lytic peptides (less than 50 amino acids) are used to construct chimeric proteins for more than one purpose. The chimeric proteins containing lytic peptides may be directly cytotoxic for the cancer cells, and/or other cells of the tumor including the tumor matrix cells and immune cells which may diminish the effects of the bacteria by eliminating them. Furthermore, the lytic peptides are useful in chimeric proteins for affecting release from the endosome. Small lytic peptides have been used in the experimental treatment of cancer. However, it is evident that most, if not all, of the commonly used antitumor small lytic peptides have strong antibacterial activity, and thus are not compatible with delivery by a bacterium (see Table 1 of Leschner and Hansel, 2004 Current Pharmaceutical Design 10: 2299-2310, the entirety of which is expressly incorporated herein by reference). Small lytic peptides useful in the technology, according to various embodiments, are those derived from Staphaloccus aureus, S. epidermidis and related species, including the phenol-soluble modulin (PSM) peptides and delta-lysin (Wang et al., 2007 Nature Medicine 13: 1510-1514, expressly incorporated herein by reference). Larger lytic peptides that may be used includes the actinoporins from sea anemones or other coelenterates, such as SrcI, FraC equinatoxin-II and sticholysin-II (Anderluh and Macek 2002, Toxicon 40: 111-124). The selection of the lytic peptide depends upon the primary purpose of the construct, which may be used in combination with other constructs providing other anticancer features. Construct designed to be directly cytotoxic to cells employ the more cytotoxic peptides, particularly PSM-.alpha.-3 and actinoporins. Constructs which are designed to use the lytic peptide to affect escape from the endosome use the peptides with the lower level of cytotoxicity, such as PSM-alpha-1, PSM-.alpha.-2 or delta-lysin.

Promoters, i.e., genetic regulatory elements that control the expression of the genes encoding the therapeutic molecules described above that are useful in the present technology, according to various embodiments, include constitutive and inducible promoters. A preferred constitutive promoter is that from the vector pTrc99a (Promega). Preferred inducible promoters include the tetracycline inducible promoter (TET promoter), SOS-response promoters responsive to DNA damaging agents such as mitomycin, alkylating agents, X-rays and ultraviolet (UV) light such as the recA promoter, colicin promoters, sulA promoters and hypoxic-inducible promoters including but not limited to the PepT promoter (Bermudes et al., WO 01/25397), the arabinose inducible promoter (AraBAD) (Los sner et al., 2007, Cell Microbiol. 9: 1529-1537; WO/2006/048344) the salicylate (aspirin) derivatives inducible promoter (Royo et al., 2007, Nature Methods 4: 937-942; WO/2005/054477), a tumor-specific promoter (Arrach et al., 2008, Cancer Research 68: 4827-4832; WO/2009/152480) or a quorum-sensing (autoinduction) promoter Anerson et al., 2006 Environmentally controlled invasion of cancer cells by engineered bacteria, J. Mol. Biol. 355: 619-627.

A single promoter may be used to drive the expression of more than one gene, such as a protease sensitive toxin and a protease inhibitor. The genes may be part of a single synthetic operon (polycistronic), or may be separate, monocystronic constructs, with separate individual promoters of the same type used to drive the expression of their respective genes. The promoters may also be of different types, with different genes expressed by different constitutive or inducible promoters. Use of two separate inducible promoter for more than one cytotoxin or other effector type peptide allows, when sufficient X-ray, tetracycline, arabinose or salicylic acid is administered following administration of the bacterial vector, their expression to occur simultaneously, sequentially, or alternatingly (i.e., repeated).

OBJECTS OF THE INVENTION

The present technology provides, according to one embodiment, live attenuated therapeutic bacterial strains that express one or more therapeutic molecules. The technology, according to various embodiments, relates specifically to certain modified forms of chimeric toxins especially suitable for expression by tumor-targeted bacteria. In a preferred embodiment, the modified toxin is derived from Pseudomonas exotoxin A (ToxA). In another preferred embodiment, the toxin is derived from cytolethal distending toxin. In a more preferred embodiment, the cytolethal distending toxin is derived from Salmonella paratyphi A, Salmonella typhi or Salmonella bongori. In particular, the technology, according to various embodiments, relates to live attenuated tumor-targeted bacterial strains that may include Salmonella sp., group B Streptococcus or Listeria vectoring chimeric anti-tumor toxins to an individual to elicit a therapeutic response against cancer. Another aspect of the technology relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, group B Streptococcus or Listeria vectoring chimeric anti-tumor toxin molecules to an individual to elicit a therapeutic response against cancer including cancer stem cells. The toxins may also be targeted to tumor matrix cells, and/or immune cells. In another embodiment of the technology, Salmonella strains including Salmonella paratyphi A, Salmonella typhi or Salmonella bongori which contain endogenous cytolethal distending toxins may, when suitably attenuated, be used as vectors for delivery of cytolethal distending toxin. In order to achieve inducible control, the endogenous reporter is replaced with an inducible promoter by homologous recombination. In another embodiment, a chimeric secreted protease inhibitor is used alone or in combination with the chimeric toxins.

Whereas the prior strains of Salmonella studied in human clinical trials used either no heterologous antitumor protein (i.e., VNP20009) or an antitumor protein located within the cytoplasm of the bacterium (i.e., cytosine deaminase expressed by TAPET-CD), or secreted proteins (Bermudes et al., WO 2001/025397) the technology, according to various embodiments, provides, according to some embodiments, methods and compositions comprising bacterial vectors that express, secrete, surface display and/or release protease inhibitors that protect co-expressed protease sensitive antitumor molecules that are also secreted, surface displayed and/or released into the tumor, lymphoma-containing lymph node, leukemic bone lumen, or proximally or topically on a carcinoma or precancerous lesion for the treatment of the neoplasia.

The primary characteristic of the bacteria of the technology, according to various embodiments, is the enhanced effect of the effector molecule such as a toxin, lytic peptide etc. relative to the parental strain of bacteria. In one embodiment, the percent increase in effect is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% greater than the parental strain of bacteria without expressing one or more protease inhibitors under the same conditions. A second characteristic of the bacteria of the technology, according to various embodiments, is that they carry novel chimeric proteins that improve their function compared to other chimeric protein expression systems. In one embodiment, the percent improvement is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of another expression system under the same conditions.

The bacteria according to a preferred embodiment of the present technology, according to various embodiments, include those modified to have little or no ability to undergo bacterial conjugation, limiting incoming and outgoing exchange of genetic material, whereas the prior art fails to limit exchange of genetic material. In addition, certain of the therapeutic molecules have co-transmission requirements (e.g., colicin proteins and colicin immunity) that are distal (i.e., genetically dissected and separated) to the therapeutic molecule location further limiting known forms of genetic exchange.

Aspects of the present technology also provide novel chimeric bacterial toxins particularly suited for expression by gram-negative bacteria. The toxins may have added targeting ligands that render them selectively cytotoxic for tumor cells, tumor stem cells and/or matrix and tumor-infiltrating immune cells. The technology also provides means to determine optimal toxin combinations which are preferably additive or more preferably synergistic. The technology also provides means to determine the optimal combination of protein toxin with conventional cancer chemotherapeutics, liposomal agents or biologics, including immunosuppressive anti-complement agents (e.g., anti-C5B). Accordingly, administration to an individual, of a live Salmonella bacterial vector, in accordance with an aspect of the present technology, that is genetically engineered to express one or more protease inhibitors as described herein co-expressed with one or more cytotoxic proteins has the ability to establish a population in the tumor, kill tumor cells, tumor stem cells as well as tumor matrix and immune infiltrating cells, resulting in a therapeutic benefit.

A preferred composition will contain, for example, a sufficient amount of live bacteria expressing the targeted cytotoxin(s) or effector proteins/peptides to produce a therapeutic response in the patient. Accordingly, the attenuated Salmonella strains described herein are both safe and useful as live bacterial vectors that can be systemically or orally administered to an individual to provide therapeutic benefit for the treatment of cancer.

Although not wishing to be bound by any particular mechanism, an effective antitumor response in humans by administration of genetically engineered, attenuated strains of Salmonella strains as described herein may be due to the ability of such mutant strains to persist within the tumor, lymphoma or leukemic bone marrow and to supply their own nutrient needs by killing tumor cells, tumor matrix and or immune infiltrating cells and further expanding the zone of the tumor that they occupy. Bacterial strains useful in accordance with a preferred aspect of the technology may carry the ability to produce a therapeutic molecule expressing plasmid or chromosomally integrated cassette that encodes and directs expression of one or more therapeutic molecules together with optionally one or more protease inhibitors, as described herein. The protease inhibitors serve to prevent the destruction of the therapeutic molecule while within the tumor. The protease inhibitor may also have an anticlotting effect, wherein a blood clot may prevent spread of the bacteria throughout the tumor. The protease inhibitor may also have direct or indirect anticancer effects through the inhibition of proteases that participate in the spread of cancerous cells. If the cytotoxin and protease inhibitor diffuse outside of the tumor, lymph node, bone lumen, proximity to a carcinoma or other neoplasia-localized distribution, they fall below the protease inhibitory concentration, no longer inhibit proteolysis of the cytotoxins or effector genes, and are then inactivated. Thus the protease inhibitor system both increases activity and provides tumor specificity.

The serovars of S. enterica that may be used as the attenuated bacterium of the live compositions described in accordance with various embodiments herein include, without limitation, Salmonella enterica serovar Typhimurium ("S. typhimurium"), Salmonella montevideo, Salmonella enterica serovar Typhi ("S. typhi"), Salmonella enterica serovar Paratyphi A ("S. paratyphi A"), Salmonella enterica serovar Paratyphi B ("S. paratyphi B"), Salmonella enterica serovar Paratyphi C ("S. paratyphi C"), Salmonella enterica serovar Hadar ("S. hadar"), Salmonella enterica serovar Enteriditis ("S. enteriditis"), Salmonella enterica serovar Kentucky ("S. kentucky"), Salmonella enterica serovar Infantis ("S. infantis"), Salmonella enterica serovar Pullorum ("S. pullorum"), Salmonella enterica serovar Gallinarum ("S. gallinarum"), Salmonella enterica serovar Muenchen ("S. muenchen"), Salmonella enterica serovar Anaturn ("S. anatum"), Salmonella enterica serovar Dublin ("S. dublin"), Salmonella enterica serovar Derby ("S. derby"), Salmonella enterica serovar Choleraesuis var. Kunzendorf ("S. cholerae kunzendorf), and Salmonella enterica serovar Minnesota (S. minnesota). A preferred serotype for the treatment of bone marrow related diseases is S. dublin. In another embodiment of the technology, Salmonella strains including Salmonella paratyphi A, Salmonella typhi or Salmonella bongori which contain endogenous cytolethal distending toxins, may, when suitably attenuated, be used as vectors for delivery of cytolethal distending toxin. In order to achieve inducible control, the endogenous reporter is replaced with an inducible promoter by homologous recombination.

By way of example, live bacteria in accordance with aspects of the technology include known strains of S. enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi) which are further modified as provided by the technology to form vectors for the prevention and/or treatment of neoplasia. Such Strains include Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, aroA-/serC-, holavax, M01ZH09, VNP20009. These strains contain defined mutations within specific serotypes of bacteria. The technology also includes the use of these same mutational combinations contained within alternate serotypes or strains in order to avoid immune reactions which may occur in subsequent administrations. In a preferred embodiment, S. Typhimurium, S. montevideo, and S. typhi which have non-overlapping O-antigen presentation (e.g., S. typhimurium is O-1, 4, 5, 12 and S. typhi is Vi, S. montevideo is O-6, 7) may be used. Thus, for example, S. typhimurium is a suitable serotype for a first injection and another serotype such as S. typhi or S. montevideo are used for a second injection and third injections. Likewise, the flagellar antigens are also selected for non-overlapping antigenicity between different injections. The flagellar antigen may be H1 or H2 or no flagellar antigen, which, when combined with the three different O-antigen serotypes, provides three completely different antigenic profiles.

Novel strains of Salmonella are also encompassed that are, for example, attenuated in virulence by mutations in a variety of metabolic and structural genes. The technology therefore may provide a live composition for treating cancer comprising a live attenuated bacterium that is a serovar of Salmonella enterica comprising an attenuating mutation in a genetic locus of the chromosome of said bacterium that attenuates virulence of said bacterium and wherein said attenuating mutation is a combinations of other known attenuating mutations. Other attenuating mutation useful in the Salmonella bacterial strains described herein may be in a genetic locus selected from the group consisting of phoP, phoQ, edt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, pabA, pts, damA, met, cys, pur, purA, purB, purl, purF, leu, ilv, arg, lys, zwf, aroA, aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB, pfkAB, crr, glk, ptsG, ptsHI, manXYZ and combinations thereof. The strain may also contain a mutation known as "Suwwan", which is an approximately 100 kB deletion between two IS200 elements. The strain may also carry a defective thioredoxin gene (trxA-; which may be used in combination with a TrxA fusion), a defective glutathione oxidoreductase (gor-) and optionally, overexpress a protein disulfide bond isomerase (DsbA). The strain may also be engineered to express invasion and/or escape genes tlyA, tlyC patI and pld from Rickettsia, whereby the bacteria exhibit enhanced invasion and/or escape from the phagolysosome (Witworth et al., 2005, Infect. Immun. 73:6668-6673), thereby enhancing the activity of the effector genes described below. The strain may also be engineered to be deleted in an avirulence (anti-virulence) gene, such as zirTS, grvA and/or pcgL, or express the E. coli lac repressor, which is also an avirulence gene in order to compensate for over-attenuation. The strain may also express SlyA, a known transcriptional activator. In a preferred embodiment, the Salmonella strains are msbB mutants (msbB-). In a more preferred embodiment, the strains are msbB- and Suwwan. In a more preferred embodiment the strains are msbB-, Suwwan and zwf-. Zwf has recently been shown to provide resistance to CO2, acidic pH and osmolarity (Karsten et al., 2009, BMC Microbiology August 18; 9:170). Use of the msbB zwf genetic combination is also particularly preferred for use in combination with administered carbogen (an oxygen carbon dioxide mixture that may enhance delivery of therapeutic agents to a tumor). In a more preferred embodiment, the strains are msbB-, Suwwan, zwf- and trxA-. In a most preferred embodiment, the strains are msbB-, Suwwan, zwf-, trxA- and gor-.

The technology also encompasses according to a preferred embodiment, gram-positive bacteria. Preferred bacteria of the technology are group B Streptococcus including S. agalaciae, and Listeria species including L. monocytogenes. It is known to those skilled in the art that minor variations in molecular biology techniques such as use of gram-positive origins of replication, gram-positive signal sequences gram-positive promoters (e.g., Lactococcus expression, Mohamadzadeh et al., PNAS Mar. 17, 2009 vol. 106 no. 11 4331-4336; Geertsma and Poolman, 2007, High-throughput cloning and expression in recalcitrant bacteria, Nature Methods 4: 705-707; Prudhomme et al., 2006, Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae, Science 313: 89-92; WO/2009/139985 Methods and materials for gastrointestinal delivery of a pathogen toxin binding agent; van Asseldonk, M et al. 1990, Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363 Gene 95, 15-160; Kim et al., J Appl Microbiol. 2008 June; 104(6):1636-43. Epub 2008 Feb. 19. Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix; Lee et al., 1999, Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins, J. Bacteriol 181(18):5790-9.) are required and substituted as needed.

Mutational backgrounds of Listeria vectors include those previously isolated, including the delta-actA strain 142 (Wallecha et al., 2009, Construction and characterization of an attenuated Listera monocytogenes strain for clinical use in cancer immunotherapy, Clin Vaccine Immunol 16: 96-103), the double D-alanine (D-ala) strain described by Jiang et al., 2007, Vaccine 16: 7470-7479, Yoshimura et al., 2006, Cancer Research 66: 1096-1104, Lenz et al., 2008, Clinical and Vaccine Immunology 15: 1414-1419, Roberts et al., 2005, Definition of genetically distinct attenuation mechanisms in naturally virulent Listeria moncyogenes by comparative cell culture and molecular characterization, Appl. Environ. Microbiol 71: 3900-3910, the actA, prfA strain by Yan et al., Infect Immun 76: 3439-3450, and those described by Portnoy et al., EP1513924 and Portnoy et al., WO/2003/102168.

Mutational backgrounds of the group B Streptococcus, S. agalactiae, include wild type (no mutations), of any of the nine serotypes that depend on the immunologic reactivity of the polysaccharide capsule and among nine serotypes, preferably types Ia, Ib, II, III, and V capable of being invasive in humans. The strain may be deleted in the beta-hemolysin/cytolysin (beta-H/C), including any member of the cly operon, preferably the clyE gene, or the CspA protease associated with virulence (Shelver and Bryan, 2008, J Bacteriol. 136: 129-134), or the hyaluronate lyse C5a peptidase CAMP factor, oligopeptidase (Liu and Nizet 2004, Frontiers in Biosci 9: 1794-1802; Doran and Nizet 2004, Mol Microbiol 54: 23-31; Herbert et al., 2004, Curr Opin Infect Dis 17: 225-229). The strains may further have mutations in metabolic genes pur, purA, aroA, aroB, aroC, aroD, pgi (glucose-6-phosphate isomerase), fructose-1,6-bisphosphatase, ptsH, ptsI, and/or one or more amino acid transporters and/or amino acid permeases. In a preferred embodiment, the strain is clyE deficient.

Other bacterial strains are also encompassed, including non-pathogenic bacteria of the gut such as E. coli strains, Bacteroides, Bifidobacterium and Bacillus, attenuated pathogenic strains of E. coli including enteropathogenic and uropathogenic isolates, Enterococcus sp. and Serratia sp. as well as attenuated Shigella sp., Yersinia sp., Streptococcus sp. and Listeria sp.

Bacteria of low pathogenic potential to humans such as Clostridium spp. and attenuated Clostridium spp., Proteus mirabilis, insect pathogenic Xenorhabdus sp., Photorhabdus sp. and human wound Photorhabdus (Xenorhabdus) are also encompassed. Probiotic strains of bacteria are also encompassed, including Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Streptococcus sp., Streptococcus agalactiae, Lactococcus sp., Bacillus sp., Bacillus natto, Bifidobacterium sp., Bacteroides sp., and Escherichia coli such as the 1917 Nissel strain.

The technology also provides, according to one embodiment, a process for preparing genetically stable therapeutic bacterial strains comprising genetically engineering the therapeutic genes of interest into a bacterially codon optimized expression sequence within a bacterial plasmid expression vector, endogenous virulence (VIR) plasmid (of Salmonella sp.), or chromosomal localization expression vector for any of the deleted genes or IS200 genes, defective phage or intergenic regions within the strain and further containing engineered restriction endonuclease sites such that the bacterially codon optimized expression gene contains subcomponents which are easily and rapidly exchangeable, and the bacterial strains so produced. Administration of the strain to the patient is therapeutic for the treatment of cancer.

The present technology provides, for example, and without limitation, live bacterial compositions that are genetically engineered to express one or more protease inhibitors combined with antitumor effector molecules for the treatment of cancers or neoplasias.

According to various embodiments, the technology provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more bacterial mutants. The technology also provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more bacterial mutants comprising nucleotide sequences encoding one or more therapeutic molecules. The pharmaceutical compositions of the technology may be used in accordance with the methods of the technology for the prophylaxis or treatment of neoplastic disease. Preferably, the bacterial mutants are attenuated by introducing one or more mutations in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway (for gram-negative bacteria), and optionally one or more mutations to auxotrophy for one or more nutrients or metabolites.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules where the therapeutic molecule is chimeric toxin.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules where the therapeutic molecule is a molecule with direct anti-cancer lytic capability.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules where the therapeutic molecule has direct anti-cancer cytotoxic or inhibitory ability.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules where the therapeutic molecule has direct anti-cellular activity against other cells of a tumor, including neutrophils, macrophages, T-cells, stromal cells, endothelial cells (tumor vasculature) and/or cancer stem cells.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules co-expressed with a protease inhibitor.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Salmonella sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated stress-resistant gram-negative bacterial mutants, wherein the attenuated stress-resistant gram-negative bacterial mutants are a Salmonella sp., and the attenuated stress-resistant gram-negative bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzymes, lytic peptides, DNAases or anti-cancer peptides.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Streptococcus sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated gram-positive bacterial mutants, wherein the attenuated gram-positive bacterial mutants are a Streptococcus sp., and the attenuated gram-positive bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzyme, lytic peptides, DNAases or anti-cancer peptides.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Listeria sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the attenuated gram-positive bacterial mutants are a Listeria sp., and the attenuated gram-positive bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzyme, lytic peptides, DNAases or anti-cancer peptides.

The present technology, according to various embodiments, encompasses treatment protocols that provide a better therapeutic effect than current existing anticancer therapies. In particular, the present technology provides methods for prophylaxis or treatment of neoplastic diseases in a subject comprising administering to said subject and one or more bacterial mutants. The present technology also provides methods for the prophylaxis or treatment of neoplastic diseases in a subject comprising administering to said subject one or more bacterial mutants, wherein said bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules together with one or more protease inhibitors.

The methods of the present technology, according to various embodiments, permit lower dosages and/or less frequent dosing of the bacterial mutants to be administered to a subject for prophylaxis or treatment of neoplastic disease to achieve a therapeutically effective amount of one or more therapeutic molecules. In a preferred embodiment, the genetically modified bacteria are used in animals, including humans, dogs, cats, and/or horses for protection or treatment against neoplastic diseases.

Accordingly, when administered to an individual, a live Salmonella, Listeria or Streptococcus bacterial vector or therapeutic, in accordance with the present technology, that is genetically engineered to express one or more anti-neoplastic molecules or molecules against other cells within the neoplastic milieu, optionally in combination with a protease inhibitor, and have improved efficacy due to improved surface display, secretion and/or released of the modified chimeric therapeutic proteins and/or enhanced binding to the target receptor resulting enhanced therapeutic activity against a neoplastic tissue including solid tumors, lymphomas and leukemias.

It is therefore an object to provide a genetic construct which causes a live host bacterium to express a peptide selected from the group consisting of at least one of: (a) a reorganized polycistronic cytolethal distending toxin fusion expressed or secreted in an absence of sty and ttsa, (b) a modified chimeric cytolethal distending toxin:apoptin fusion expressed or secreted in an absence of in an absence of sty and ttsa, and (c) a functionally active modified chimeric Pseudomonas ToxA therapeutic molecule. The genetic construct or bacterium may be provided in a pharmaceutically acceptable dosage form, suitable for administration to a human or animal, without causing significant morbidity. The peptide may act as an antineoplastic agent, and the bacterium may be trophic for diseased or malignant growths. The dosage form may be oral, enteral, parenteral, intravenous, per anus, topical, or inhaled, for example. The peptide may comprise a combination of at least one secretion signal, a linker, and domain Ib.

A pharmaceutically effective dosage form may comprise between about 10.sup.5 to 10.sup.12 live bacteria, within a lyophilized medium for oral administration. In some embodiments, about 10.sup.9 live bacteria are administered.

The live host bacterium may have antineoplastic activity against lymphoma, or solid tumors.

The peptide may be, for example, the modified chimeric cytolethal distending toxin:apoptin fusion with antineoplastic activity, having deletions of at least one cytolethal distending toxin nuclear localization signal. The peptide may also comprises an N-terminal fusion, e.g., of the modified chimeric cytolethal distending toxin:apoptin fusion.

Another object of the technology provides a chimeric protease inhibitor comprising YebF fused to sunflower trypsin inhibitor, adapted to inhibit at least one serine protease. The chimeric protease inhibitor may be formed by a genetically engineered bacteria, wherein the genetically engineered bacteria secretes the YebF fused to sunflower trypsin inhibitor. The chimeric protease inhibitor may be provided in combination with a host bacteria and a genetically engineered construct which encodes the chimeric protease inhibitor, wherein the host bacteria secretes the chimeric protease inhibitor and the chimeric protease inhibitor inhibits at least one serine protease.

A further object provides a method for treating neoplastic tumor cells within a living organism, comprising: administering to a human or animal a live genetically engineered bacterium expressing peptide selected from the group consisting of at least one of: (a) a reorganized polycistronic cytolethal distending toxin fusion with antineoplastic activity expressed or secreted in an absence of sty and ttsa, (b) a modified chimeric cytolethal distending toxin:apoptin fusion with antineoplastic activity expressed or secreted in an absence of in an absence of sty and ttsa, and (c) a functionally active modified chimeric Pseudomonas ToxA therapeutic molecule with antineoplastic activity, wherein said peptide is secreted or released by said bacterium in a functionally active form. According to one embodiment of the method, the live genetically bacterium is allowed to colonize at least one tissue of the human or animal after administration, and the human or animal is treated with at least one systemic antibiotic to which the live genetically engineered bacterium is sensitive. The antibiotic may be narrow spectrum, and indeed, may comprise a composition not generally toxic to other bacteria or animal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I shows a comparison of modified ToxA chimeras.

FIGS. 2A-2E show C-terminal modified cldt from Salmonella paratyphi A.

FIG. 3A-3D show N-terminal modified cldt from Salmonella paratyphi A.

FIGS. 4A-4D show nuclear localization signal (NLS) modified cldt from Salmonella paratyphi A.

FIGS. 5A-5D show a secreted protease inhibition assay.

FIG. 6 shows a pAra-Txa10 4,678 bp plasmid.

FIGS. 7A and 7B show results of MTT cytotoxicity (anticancer cell killing) assay of protease challenge/protease inhibitors.

FIGS. 8A and 8B show results of a cytotoxicity (anticancer cell killing) assay of cytolethal distending toxin and cytolethal distending toxin:apoptin fusions.

FIG. 9 shows results of an Azocoll protease inhibition assay.

FIGS. 10A and 10B show a cytotoxicity (anticancer cell killing) assay of Exotoxin A (ToxA) and chimeric TGF.alpha.-ToxA fusions.

FIGS. 11A and 11B shows a pTet-1 3,156 plasmid, and pTet-1 under tetracycline controlled transcriptional activation.

FIG. 12 shows a flowchart of a method for detection of protease inhibitory activity on protein based plates.

FIG. 13 shows Dinitrosalicylic Acid (DNS) assay for the detection of reducing sugars produced by active amylase under a trypsin challenge.

DETAILED DESCRIPTION OF THE INVENTION

The present technology provides, according to various embodiments, live attenuated therapeutic bacterial strains that express one or more therapeutic with improved expression, secretion, surface display and/or release and/or have improved binding and anticancer cell activity that results in improved therapeutic efficacy. In particular, one aspect of the technology relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, Streptococcus or Listeria vectoring novel chimeric anti-tumor toxins to an individual to elicit a therapeutic response against cancer. The types of cancer may generally include solid tumors, carcinomas, leukemias, lymphomas and multiple myelomas. Another aspect of the technology relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, Streptococcus, Clostridium and Listeria that encode anti-neoplastic molecules to an individual to elicit a therapeutic response against cancers including cancer stem cells, immune infiltrating cells and or tumor matrix cells.

For reasons of clarity, the detailed description is divided into the following subsections: targeting ligands; chimeric bacterial toxins; and secreted protease inhibitors.

Targeting Ligands

Targeting ligands have specificity for the target cell and are used to both confer specificity to chimeric proteins, and to direct attachment and/or internalization into the target cell. The ligands are known ligands or may be novel ligands isolated through standard means such as phage display (Barbass III et al., 2004, Phage Display, A Laboratory Manual, Cold Spring Harbor Press) including the use of commercially available kits (Ph.D-7 Phage Display Library Kit, New England Biolabs, Ipswich, Mass.; Li et al., 2006. Molecular addresses of tumors: selection by in vivo phage display. Arch Immunol Ther Exp 54: 177-181,). The ligands of various aspects of the present technology are peptides that can be expressed as fusions with other bacterially-expressed proteins. The peptides may be further modified, as for gastrin and bombesin, in being amidated by a peptidylglycine-alpha-amidating monoxygenase or C-terminal amidating enzyme, which is co-expressed in the bacteria that use these peptides using standard molecular genetic techniques. Examples of targeting peptides are shown in Bermudes U.S. Pat. No. 8,524,220 Table 4, incorporated by reference herein. These ligands and their targets include TGF-.alpha. (EGFR), HAVDI and INPISGQ and dimeric versions (N-cadherin of prostate), DUP-1 peptide (prostate cancer), laminin-411 binding peptides (brain neovasculature), pertussis toxin S3 subunit (cancer cells), DARPINS (e.g., H10, HER2), affibody against Her2 (Zielenski, R., Lyakhov, I., Jacobs, A., Chertov, O., Kramer-Marek, G., Francella, N., Stephen, A., Fisher, R., Blumenthal, R., and Capala, J. Affitoxin--A Novel Recombinant, HER2-Specific, Anti-Cancer Agent for Targeted Therapy of HER2-Positive Tumors. J Immunother. 2009 October; 32(8):817-825) luteinizing hormone-releasing hormone (LHRH receptor), IL2 (IL2R), EGF and EGF receptor related peptide (EGFR), tissue factor (TfR), IL4 (IL4R), IL134 (IL13R), GM-CSF (GM-CSFR), CAYHRLRRC SEQ ID NO.: 021 (lymphoid tissue; AML), A33 antigen binding peptide (A33) CLTA-4/CD152 melanoma, CD19 binding peptides/Bpep (alpha(v) beta(6) integrin (.alpha.v.beta.6), non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL)), CD20 binding peptides (CD20, B-cell malignancies), CD22 binding peptides (B lymphocytes, hairy cell leukemia), CD25 binding peptides (chemotherapy-resistant human leukemia stem cells), TRU-015 (CD-20), CD30 binding peptides (CD-30 Hodgkin's lymphoma), CD32 binding peptides (chemotherapy resistant human leukemia stem cells), CD33 binding peptides (CD-33 AML myleodysplastic cells MDS)), CD37 binding peptides (leukemia and lymphoma), CD40 binding peptides (CD40 multiple myeloma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), Hodgkin lymphoma and acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, refractory non-Hodgkin lymphoma, including follicular lymphoma), CD52 (CLL), CD55 (CD55R), CD70 (hematological malignancies, non-Hodgkin's lymphoma), CD123 binding peptides (AML), RGD peptides (tumor cells and tumor endothelium), nanobodies derived from camels and llamas (camelids), including humanized nanobodies and VHH recognition domains (cancer), bombesin (gastrin releasing peptide receptor), gastrin releasing peptide (gastrin releasing peptide receptor), somatostatin octapeptide RC-121 (colon cancer), vasoactive intestinal peptide (tumor cell membranes), PTHrP (parathyroid hormone receptor G-protein coupled receptor), mesothelin binding peptides (mesothelin), CA125/MUC16 (mesothelin), heat stable enterotoxin (HST) (guanylyl cyclase C), GM-CSF (AML), vitronectin (Alfa(V)Beta(3) integrin), gastrin (gastrin receptor), CQTIDGKKYYFN SEQ ID NO.: 022 peptide from Clostridium, affibody against HER3, DARPIN against HER2, TGF.alpha., EGF, EGFR-binding peptides and other, non-limiting, peptides. In preferred embodiments, the peptides are affibody against HER2, H10 DARPIN against HER2, TGF.alpha., EGF, EGFR-binding peptides.

FIGS. 1A-1I show a comparison of modified ToxA chimeras. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added to any of the constructs.

FIG. 1A shows a chimera containing an OmpA secretion signal, TGF .alpha. targeting domain, a GGGGS(.times.3) (i.e., GGGGSGGGGSGGGGS, SEQ ID NO.: 008 linker, ToxA domain Tb (containing cysteine bonding), ToxA domain III, followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023).

FIG. 1B shows a chimera containing an OmpA secretion signal, TGF.alpha. targeting domain, a GGGGS(.times.3), SEQ ID NO.: 008 linker), ToxA domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added.

FIG. 1C shows a chimera containing an OmpA secretion signal, TGF.alpha. targeting domain, a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added.

FIG. 1D shows a chimera containing an OmpA secretion signal, EGF binding peptide (EGB), a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added.

FIG. 1E shows a chimera containing an M13 pIII secretion signal, EGF binding peptide (EGB), a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added.

FIG. 1F shows a chimera containing an OmpA secretion signal, Her2 affibody binding, a GGGGS linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

FIG. 1G shows a chimera containing an OmpA secretion signal, Her2 affibody binding, a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

FIG. 1H shows a chimera containing an OmpA secretion signal, Her2 affibody, a GGGGS(.times.3) linker, ToxA domain Ib (containing cysteine bonding), ToxA domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

FIG. 1I shows a chimera containing an OmpA secretion signal, EGFR binding peptide, a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA domain Ib (containing cysteine bonding), ToxA domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

Chimeric Bacterial Toxins

Chimeric toxins are toxins that may contain combinations of elements including targeting peptides, flexible linkers, disulfide bonding, lytic peptides, nuclear localization signals, blocking peptides, protease cleavage (deactivation or activation) sites, N- or C-terminal secretion signals, autotransporter constructs, used to adapt the proteins to be expressed, secreted, surface displayed and/or released by bacteria to provide therapeutic molecules that are effective in treating neoplastic cells, stromal cells, neoplastic stem cells as well as immune infiltrating cells. Targeting to a particular cell type uses the appropriate ligand described above or from other known sources. Toxin activity is determined using standard methods known to those skilled in the art such as Aktories (ed) 1997 (Bacterial Toxins, Tools In Cell Biology and Pharmacology, Laboratory Companion, Chapman & Hall).

FIGS. 2A-2D show C-terminal fusions of modified cldt from Salmonella paratyphi A.

FIG. 2A shows the Salmonella typhoid toxin cytolethal distending toxin subunit B (cldtB) together with sty, ttsA, pltA and pltB. The entire operon is with cldtB and sty under control of an inducible promoter such as the arabinose inducible promoter, with pltB, pltA and ttsA under control of their upstream region.

FIG. 2B shows the Salmonella typhoid toxin cytolethal distending toxin subunit B (cldtB) together with pltB and pltA as a single artificial operon under control of an inducible promoter, and the genes sty and ttsA removed.

FIG. 2C shows the cldtB, pltB and pltA artificial operon with an in frame fusion of the restriction enzymes SexAl, HindIII and Xbal followed by a stop codon.

FIG. 2D shows the cldtB, pltB and pltA artificial operon with SexAl, HindIII and Xbal with the FLAG epitope and a GGGGS(.times.3), SEQ ID NO.: 008 linker inserted in the SexAl and HindIII sites.

FIG. 2E shows the cldtB, pltB and pltA artificial operon with SexAl, HindIII and Xbal with the FLAG epitope and a GGGGS(.times.3), SEQ ID NO.: 008 linker inserted in the SexAl and HindIII sites and apoptin fragments inserted in-frame into the HindIII and Xbal sites. The apoptin and/or apoptin fragments can consist of 1) apoptin 1-121, 2) apoptin 33-121, 3) apoptin 82-121, 4) apoptin 97-121, 5) apoptin 106-121, 6) apoptin 111-121 or 7) apoptin 1-31 linked ot 83-121 or 8) TAT-apoptin.

Chimeric ToxA Forms Secreted by Bacteria.

Cytotoxic forms of ToxA that can be secreted by bacteria have targeting ligands. Previous ToxA conjugates were not adapted for secretion by bacteria in an active form. For example, TGF.alpha.-GIII-KDEL (Kihara and Pastan 1994, Small Chimeric Toxins Containing Only Transforming Growth Factor .alpha. and Domain III of Pseudomonas Exotoxin with Good Antitumor Activity in Mice. Cancer Res 1994:54:5154-5159) had a TGF.alpha. .alpha.-PE38 which has a deletion of amino acids 365-380 of domain I (Kreitman et al., 1992, Properties of Chimeric Toxins with Two Recognition Domains: Interleukin 6 and Transforming Growth Factor .alpha. at Different Locations in Pseudomonas Exotoxin. Bioconjugate Chem. 3, 63-68) have no signal peptide, only a single GGGGS linker with 10 amino acids upstream of DM, compared to the present version (FIGS. 1A-1I). PE38-KDEL (Kreitman et al., 1994, Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin 2 receptor-expressing human carcinoma. Blood 1994, 83, 426-434) uses anti-Tac (Fv) as binding ligand rather than those we describe here. Similarly, the Affitoxin (Zielenski et al., 2009, Affitoxin--A Novel Recombinant, HER2-Specific, Anti-Cancer Agent for Targeted Therapy of HER2-Positive Tumors. J Immunother 32: 817-825) has no signal peptide and only a short flexible linker. The forms of ToxA shown in FIGS. 1A-1I and described in the examples with targeting moieties supply the necessary components for secreted by bacteria and are cytotoxic for cancer cells.

FIGS. 3A-3D show N-terminal fusions of modified cldt from Salmonella paratyphi A.

FIG. 3A shows the Salmonella typhoid toxin cytolethal distending toxin subunit B (cldtB) together with pltB and pltA as a single artificial operon under control of an inducible promoter, and the genes sty and ttsA removed. The presence and relative location of an N-terminal signal sequence (SS) is shown.

FIG. 3B shows the cldtB, pltB and pltA artificial operon with an in frame fusion of the restriction enzymes HindIII, Xbal and Clal inserted after the signal sequence.

FIG. 3C shows the cldtB, pltB and pltA artificial operon with HindIII, Xbal and Clal with the FLAG epitope and a GGGGS(.times.3), SEQ ID NO.: 008 linker inserted in the Xbal and Clal sites.

FIG. 3D shows the cldtB, pltB and pltA artificial operon with the FLAG epitope and a GGGGS(.times.3), SEQ ID NO.: 008 linker inserted in the Xbal and Clal sites and apoptin fragments inserted in-frame into the HindIII and Xbal sites. The apoptin and/or apoptin fragments can consist of 1) apoptin 1-121, 2) apoptin 33-121, 3) apoptin 82-121, 4) apoptin 97-121, 5) apoptin 106-121, 6) apoptin 111-121 or 7) apoptin 1-31 linked to 83-121 or 8) TAT-apoptin.

Chimeric Cytolethal Distending Toxins.

Cytolethal distending toxins (cldt) including those cldts from Haemophilus, Aggregatibacter, Salmonella, Escherichia, Shigella, Campylobacter, Helicobacter, Hahella and Yersinia, typhoid toxins (pertussis like toxin) (pltAB), pertussis toxin, cldt:plt hybrids are three component toxins of these bacteria. Cldt is an endonuclease toxin and has a nuclear localization signal on the B subunit. Chimeric toxins are provided that utilize N-terminal or C-terminal fusions to apoptin, a canary virus protein that has a tumor-specific nuclear localization signal, and a normal (non-transformed) cell nuclear export signal (FIGS. 2A-4D). The present technology, according to one embodiment, consists of a modified Salmonella CLDT operon and forms of cytolethal distending toxins that are chimeric with apoptin and other peptide moieties including peptide linkers that improve activity and peptide blocking moieties that must be specifically cleaved to activate the protein.

The present technology, according to one embodiment. uses deletions in the CLDT nuclear localization signals which are then complemented by N- or C-terminal fusions with apoptin, or apoptin fragments, which supply its nuclear localization signal in trans as a fusion peptide.

The cytolethal distending toxin B and chimeric cltdB may be expressed as a polycistronic construct consisting of cldtABC. The cytolethal distending toxin B and chimeric cltdB may be expressed as a polycistronic construct consisting containing the typhoid pertussis-like toxin (plt) AB genes. However, in the present technology, according to one embodiment, the presence of sty and ttsA are not required for secretion of the active toxin when the operon is reorganized into a unidirectional operon of cldtB, pltB and pltA.

Overall improvement is defined as an increase in effect, such as the ability to kill a neoplastic cells in vitro by the bacteria, or inhibit or reduce the volume or cell number of a solid tumor, carcinoma, lymphoma or leukemia in vivo following administration with the bacteria expressing a therapeutic molecule, with and without the protease inhibitor. The effect of the protein therapeutic activity is determined using standard techniques and assays known to those skilled in the art. The contribution of the therapeutic protein and protease inhibitors is determined individually and in combination. Additivity, synergy or antagonism may be determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods.

FIGS. 4A-4D show nuclear localization signal (NLS) modified partially or completely cldt from Salmonella paratyphi A.

FIG. 4A shows inactivated Salmonella paratyphi A typhoid toxin as a delivery mechanism for peptide fusions. The Salmonella typhoid toxin cldtB is inactivated by site-directed mutagenesis of the known active sites such as H160Q and/or H259Q. Fusions to the inactivated form retain the secretion and delivery to the cytosol and allow transport to the nucleus.

FIG. 4B shows inactivated Salmonella paratyphi A typhoid toxin nuclear localization signals (NLS1) enhancing dependence on apoptin NLSs and/or nuclear export as a mode of tumor cell-specific delivery of an active cldtB.

FIG. 4C shows inactivation of NLS2 and

FIG. 4D shows both portions of the cldtB bipartite nuclear localization signals can be altered from charged (basic) e.g., arginines and/or histidines to polar serines or threonines.

Secreted Protease Inhibitors

A chimeric protease inhibitor secreted by Escherichia coli with the potential for use in biofuel fermentations and therapeutic bacteria is provided.

There is growing interest in the use of live bacteria targeting solid tumors as an alternative to or in conjunction with standard chemotherapy and radiation treatment. These bacteria can be engineered to secrete bacterial derived cytotoxins, such as Pseudomonas aeruginosa exotoxin A, directly to the site of the tumor (Forbes, 2010 and Swofford et al, 2014). Therapeutic bacteria expressing and secreting cytotoxins that are tumor cell specific may enhance tumor destruction and spare healthy adjacent tissues (Siegal et al, 1989). Bacteria expressing protease inhibitors specific for proteases associated with metastasis is an innovative approach to cancer therapeutics. Co-expression of a tumor specific cytotoxin and a protease inhibitor may enhance the stability of the toxin by protecting it in the highly proteolytic environment of the tumor while confining the cancer.

From a bio-industrial standpoint, fermentations which are sensitive to bacterial contaminations, especially those due to proteolytic strains such as Lactobacillus sp., may benefit from protease inhibition. Bacteria and fungi secreting protease inhibitors offer a potential augmentation to extracellular fermentations such as cellulosic ones used in the production of biofuels where microbial contaminations reduce production efficiency.

Protease inhibitor-secreting bacteria have an ability to protect extracellular proteins from proteolytic destruction by trypsin, utilizing exotoxin A in cytotoxicity studies and .alpha.-amylase as a model industrial extracellular fermentation enzyme.

FIGS. 5A-5D show results of a secreted protease inhibition assay for detection of bacterial secreted protease inhibitors, using the method of Quintero and Bermudes (2014. A culture-based method for determining the production of secreted protease inhibitors. J. Micro. Methods. 100, 105-110).

FIG. 5A shows growth pattern of E. coli harboring 1) pTet-1 (EV), 2) pTetOmpA-SFTI, 3) pTetTxA-SFTI, 4) pTetOmpA-SFTI-RBS Lys, 5) pTetTxA-SFTI-RBS Lys on BCG 0.5% Casein induced with 1 .mu.g/mL anhydrotetraclyine HCl after 24 hours at 37.degree. C.

FIG. 5B shows staining pattern of protected protein, using the method of Quintero and Bermudes (2014. A culture-based method for determining the production of secreted protease inhibitors. J. Micro. Methods. 100, 105-110).

FIG. 5C shows growth of E. coli harboring 1) pTet-1 (EV), 2) pTetYebF, 3) pTetYebF-SFTI, on BCG 0.5% Casein with 1 mg/mL anhydrotetraclyine HCl after 24 hours at 37.degree. C.

FIG. 5D shows a staining pattern of protected protein.

FIG. 6 shows the gene encoding exotoxin A, which was amplified from Pseudomonas aeruginosa str. PA103 in a colony PCR and ligated to pAra-1 to create pAra-Txa10. The plasmids were electroporated into Salmonella typhimurium str. JR501 and induced with 0.1% arabinose. Filter sterile supernatant was diluted 2-fold in PBS. MDA-MB-468 breast cancer cells were plated at 2.5.times.103 cells in DMEM with 10% FBS and allowed to adhere to a 96-well plate for 24 hours. Bacterial supernatant or PBS was delivered to the cell culture to 10% and allowed to incubate for an additional 72 hours.

The sunflower trypsin inhibitor (SFTI), a 14 amino acid cyclic peptide was chosen for its small size and ability to inhibit matriptase, a tumor associated protease over-expressed in cancers of the breast, ovaries, prostate and liver. (Cai et al, 2011). SFTI was fused to the secretion signal peptides derived from the outer membrane protein OmpA and the secreted cytotoxin exotoxin A and evaluated for its ability to be secreted extracellularly and to inhibit degradation of a protein substrate. These constructs were coupled with the pColE3 lysis protein, which activates phospholipase and outer-membrane porins to facilitate secretion. SFTI was also fused to YebF, which is known to accumulate in the extracellular milieu of select proteobacteria.

Therefore, it can be seen that SFTI can be secreted in low amounts when fused to a type II secretion signal peptide. Co-expression of pColE3 lysis protein with the fusions enhances secretion when the OmpA signal is utilized, but not the Exotoxin A signal. This may be due to ineffective cleavage of the latter in the periplasm. Fusion of SFTI to YebF demonstrated high protease inhibition both in solid and liquid culture media. We were able to show the protective effect of YebF-SFTI by conducting an in vitro digest of Exotoxin A followed by a cytotoxity assay, which demonstrated sustained toxin activity. An in vitro digest of amylase followed by DNS assay showed only slight inhibition of trypsin by YebF-SFTI for unknown reasons.

FIG. 7A shows cytotoxicity (anticancer cell killing) protease challenge/protease inhibitor assay results, in particular protection of extracellular ToxA from trypsin by YebF-SFTI assessed by cell killing. MDA-MB-468 breast cancer cells assessed by MTT after supernatants from S. typhimurium harboring pAra-1 was mixed with supernatants from E. coli harboring pTet-1 (EV), pTetYebF, or pTetYebf-SFTI then delivered to the cell culture. A 1:4 (vol:vol) mixture of supernatant containing exotoxin A and either YebF or YebF-SFTI was prepared and delivered to the cell culture. A seven hour in vitro digest (IVD) at 37.degree. C. utilizing trypsin at a final concentration of 0.1 mg/ml containing exotoxin A and either YebF or YebF-SFTI (1:4) was conducted to test if the secreted protein maintained measurable activity.

FIG. 7B shows protection of extracellular ToxA from trypsin by YebF-SFTI assessed by cell killing. MDA-MB-468 breast cancer cells were plated as above, and assessed by MTT after Supernatants from S. typhimurium harboring pAra-1 was mixed with supernatants from E. coli harboring pTet-1 (EV), pTetYebF, or pTetYebf-SFTI then delivered to the cell culture. A 1:4 (vol:vol) mixture of supernatant containing exotoxin A and either YebF or YebF-SFTI was prepared and delivered to the cell culture. A seven hour in vitro digest (IVD) at 37.degree. C. utilizing trypsin at a final concentration of 0.1 mg/ml containing exotoxin A and either YebF or YebF-SFTI (1:4) was conducted to test if the secreted protein maintained measurable activity.

See: Forbes, N. S., 2010. Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer 10, 785-794; Swofford, C., St. Jean, A. T., Panteli, J. T., Brentzel, Z. J., Forbes, N. S. 2014. Identification of Staphylococcus aureus .alpha.-hemolysin as a Protein Drug that is Secreted by Anticancer Bacteria and Rapidly kills Cancer Cells. Biotech. Bioeng. 111, 1233-1245; Siegall, C., Yong-Hua, X., Vijay, K. C., Sankar, A., Fitzgerald, D., and Pastin, I. 1989. Cytotoxic activities of a fusion protein comprised of TGF.alpha. and Pseudomonas exotoxin. FASEB. J. 3, 2547-2652; Quintero, D. and Bermudes, D. 2014. A culture-based method for determining the production of secreted protease inhibitors. J. Micro. Methods. 100, 105-110.

See also Examples 12-14.

FIGS. 8A and 8B show results of a cytotoxicity (anticancer cell killing) assay of Salmonella paratyphi A cytolethal distending toxin and cytolethal distending toxin:apoptin fusions toward MDA-MB-468 breast cancer cells.

NT: no treatment.

PBS: phosphate buffered saline.

1: treatment with 10 .mu.L of a bacterial culture supernatant carrying an empty vector plasmid into a 100 .mu.L tissue culture wells, with dilutions of 1, 1/2, 1/4, 1/8 and 1/16.

2: the cloned wild type S. paratyphi A cytolethal distending operon (as shown in FIGS. 2A-2E), with dilutions of 1/2, 1/4, 1/8 and 1/16.

3: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus with dilutions of 1/2, 1/4, 1/8, and 1/16.

4: the reorganized polycistronic S. paratyphi A cytolethal distending operon without a polylinker in cldtB C-terminus with dilutions of 1/2 1/4 1/8 and 1/16. Absorbance at 570 nm indicates the relative number of live cells.

5: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a flexible linker GGGGS(3) SEQ ID NO.: 008 with dilutions of 1/2, 1/4, 1/8, and 1/16.

6: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of apoptin 1-121 with dilutions of 1/2, 1/4, 1/8, and 1/16.

7: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of apoptin 82-121 with dilutions of 1/2, 1/4, 1/8 and 1/16.

8: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of apoptin 111-121 with dilutions of 1/2, 1/4, 1/8 and 1/16.

9: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of apoptin 97-121 with dilutions of 1/2, 1/4, 1/8 and 1/16.

10: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of apoptin 33-121 with dilutions of 1/2, 1/4, 1/8 and 1/16.

11: the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus and a fusion of TAT:apoptin 1-121 with dilutions of 1/2, 1/4, 1/8 and 1/16.

FIG. 9 shows results of an assay using Azo dye impregnated collagen (Azocoll) protease substrate. E. coli harboring pTet-1 (EV), pTetYebF, or pTetYebF-SFTI, were induced overnight. 500 .mu.L of filter sterile supernatant was incubated with 10 mL of 6.25 mg/mL trypsin solution for 1 hour at room temperature to allow binding. Soybean inhibitor (SBI) and LB served as controls. After equilibration, 400 mL of a 1.5 mg/mL Azocoll suspension was mixed with each sample and incubated at 37.degree. C. for a 2 hour digestion. Proteolytic degradation of Azocoll was measured with a spectrophotometer at Abs 550 nm. After subtracting background, the absorbance values were plotted using Prism version 6.

FIGS. 10A and 10B show the cytotoxicity of Pseudomonas aeruginosa Exotoxin A (ToxA) and chimeric derivatives toward MDA-MB-468 breast cancer cells.

FIG. 10A:

NT: no treatment. PBS, phosphate buffered saline.

EV 1: treatment with 10 .mu.L of a bacterial culture supernatant carrying an empty vector plasmid into a 100 .mu.L tissue culture wells.

1: the cloned wild type Pseudomonas aeruginosa Exotoxin A (ToxA) with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

2: A chimeric toxin composed of N-terminal TGF.alpha. fused to domains Ib and III of ToxA with a C-terminal KDEL sequence with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

3: A chimeric toxin composed of N-terminal TGF.alpha. fused to domains Ib and III of ToxA with a C-terminal KDEL sequence co-expressed with the RBS E3 Lysis protein with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

4: A chimeric toxin composed of N-terminal TGF.alpha. fused to domains Ib and III of ToxA with the native C-terminal REDLK SEQ ID NO.: 023 ToxA sequence with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

EV 2: empty vector plasmid.

FIG. 10B:

NT: no treatment. PBS, phosphate buffered saline.

EV 1: treatment with 10 .mu.L of a bacterial culture supernatant carrying an empty vector plasmid into a 100 .mu.L tissue culture wells.

1: the cloned wild type Pseudomonas aeruginosa Exotoxin A (ToxA) with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

2: A chimeric toxin composed of N-terminal TGF.alpha. fused to domain III of ToxA with a C-terminal KDEL sequence with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

3: A chimeric toxin composed of N-terminal TGF.alpha. fused to domain III of ToxA with a C-terminal KDEL sequence co-expressed with the RBS E3 Lysis protein with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

4: A chimeric toxin composed of N-terminal TGF.alpha. fused to domains Ib and III of ToxA with the native C-terminal REDLK SEQ ID NO.: 023 ToxA sequence with dilutions of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64.

EV 2: empty vector plasmid.

FIGS. 11A and 11B show pTet-1 under tetracycline controlled transcriptional activation (FIG. 11A), and insertion sequences downstream from the tetracycline repressor sequence (FIG. 11B). The sunflower trypsin inhibitor DNA sequence was fused to the signal peptide sequences derived from OmpA and Exotoxin A and ligated to pTet-1. The pColE3 lysis sequence, including its Shine-Delgarno sequence was cloned into pTetOmpA-SFTI and pTetTxA-SFTI. The YebF sequence was cloned into pTet-1 to create pTet-YebF. The SFTI sequence was fused in frame with YebF to produce pTetYebF-SFTI. All constructs were verified by sequencing then transformed into electrocompetent E. coli str. JW2197-1 lacking the periplasmic trypsin inhibitor, ecotin.

FIG. 12 shows a flowchart of a method for detection of protease inhibitory activity on protein based plates.

FIG. 13 shows Dinitrosalicylic Acid (DNS) assay for the detection of reducing sugars produced by active amylase under a trypsin challenge. 200 mL of the YebF and YebF-SFTI supernatants or LB was mixed with 10 mL of a 1.25 mg/mL trypsin solution and allowed to equilibrate for 30 minutes. Heat inactivated trypsin served as a control. 10 mL amylase at 1 mg/mL was considered a 1.times. concentration and two-fold dilutions to 1/16.times. were conducted. The amylase was then added to the mixtures and incubated at 37.degree. C. for five hours to allow digestion. 100 mL of 1% starch solution was then added and the reaction allowed to proceed for fifteen minutes. DNS was added and the mixture was boiled for 5 minutes, diluted with 500 mL H2O, and absorbance was read at 515 nm.

EXAMPLES

In order to more fully illustrate the technology, the following examples are provided.

Example 1

A Salmonella expression vector.

Inducible expression vectors for E. coli and Salmonella, such as arabinose inducible expression vectors, are widely available and known to those skilled in the art. By way of example, an expression vector typically contains a promoter which functions to generate an mRNA from the DNA, such as an inducible arabinose promoter with a functional ribosomal binding site (RBS) an initiation codon (ATG) and suitable cloning sites for operable insertion of the functional DNA encoding the effector proteins described below into the vector, followed by a transcriptional termination site, plasmid origin of replication, and an antibiotic resistance factor that allows selection for the plasmid. Vectors that lack antibiotic resistance such as asd(-) balanced lethal vectors (Galan et al., 1990 cloning and characterization of the asd gene of Salmonella Typhimurium: use in stable maintenance of recombinant Salmonella vaccine strains, Gene 94: 29-35) may also be used, or insertion into the chromosome.

Example 2

Modified TGF.alpha.-GIII-KDEL (A. Kihara and I. Pastan. Small Chimeric Toxins Containing Only Transforming Growth Factor .alpha. and Domain III of Pseudomonas Exotoxin with Good Antitumor Activity in Mice. Cancer Res 1994:54:5154-5159). A modified form with an added OmpA signal sequence, extended flexible linker and presence of domain Ib with enhanced activity functionally expressed, secreted, surface displayed and/or released is shown in FIG. 1A. The construct contains an OmpA secretion signal, TGF.alpha. targeting domain, a GGGGS(.times.3) SEQ ID NO.: 008 linker, ToxA domain Ib (containing cysteine bonding), ToxA domain III followed by, optionally KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023 is used. The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1364 is:

TABLE-US-00001 SEQ ID NO.: 001 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTctgacctgcccggtcgcc gccggtgaatgcgcgggcccggcggacagcggcgacgccctgctggagcg caactatcccactggcgcggagttcctcggcgacggcggcgacgtcagct tcagcacccgcggcacgcagaactggacggtggagcggctgctccaggcg caccgccaactggaggagcgcggctatgtgttcgtcggctaccacggcac cttcctcgaagcggcgcaaagcatcgtcttcggcggggtgcgcgcgcgca gccaggacctcgacgcgatctggcgcggtttctatatcgccggcgatccg gcgctggcctacggctacgcccaggaccaggaacccgacgcacgcggccg gatccgcaacggtgccctgctgcgggtctatgtgccgcgctcgagcctgc cgggcttctaccgcaccagcctgaccctggccgcgccggaggcggcgggc gaggtcgaacggctgatcggccatccgctgccgctgcgcctggacgccat caccggccccgaggaggaaggcgggcgcctggagaccattctcggctggc cgctggccgagcgcaccgtggtgattccctcggcgatccccaccgacccg cgcaacgtcggcggcgacctcgacccgtccagcatccccgacaaggaaca ggcgatcagcgccctgccggactacgccagccagcccggcaaaccgccga aagatgaactgtaaCTCGAGTagatctgtactCTAGAGTCGACCTGCAGG CATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATA CAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGG CGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGA AACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGG AACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCT TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT CCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCG GGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCA TCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTT TCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG TGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG TTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGC TATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCAC AGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAA CTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC AGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCC CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTC TGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTG AGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGAC TTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT TGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTA TTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAG CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTT TCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1364 together with the colicin E3 lysis protein from 1399 to 1542 is:

TABLE-US-00002 SEQ ID NO.: 002 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTctgacctgcccggtcgcc gccggtgaatgcgcgggcccggcggacagcggcgacgccctgctggagcg caactatcccactggcgcggagttcctcggcgacggcggcgacgtcagct tcagcacccgcggcacgcagaactggacggtggagcggctgctccaggcg caccgccaactggaggagcgcggctatgtgttcgtcggctaccacggcac cttcctcgaagcggcgcaaagcatcgtcttcggcggggtgcgcgcgcgca gccaggacctcgacgcgatctggcgcggtttctatatcgccggcgatccg gcgctggcctacggctacgcccaggaccaggaacccgacgcacgcggccg gatccgcaacggtgccctgctgcgggtctatgtgccgcgctcgagcctgc cgggcttctaccgcaccagcctgaccctggccgcgccggaggcggcgggc gaggtcgaacggctgatcggccatccgctgccgctgcgcctggacgccat caccggccccgaggaggaaggcgggcgcctggagaccattctcggctggc cgctggccgagcgcaccgtggtgattccctcggcgatccccaccgacccg cgcaacgtcggcggcgacctcgacccgtccagcatccccgacaaggaaca ggcgatcagcgccctgccggactacgccagccagcccggcaaaccgccga aagatgaactgtaaCTCGAGTagatctgtactCTAGAAAGGAGTCGTTAT GAAAAAAATAACAGGGATTATTTTATTGCTTCTTGCAGTCATTATTCTGT CTGCATGTCAGGCAAACTATATCCGGGATGTTCAGGGCGGGACCGTATCT CCGTCATCAACAGCTGAAGTGACCGGATTAGCAACGCAGTAACTGCAGGC ATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATAC AGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGC GGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAA ACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGA ACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATC CGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGG GCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCAT CCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAA TGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGT GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT ATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAAC TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC CGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT TACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 3

Modified TGF.alpha.-GIII-KDEL (A. Kihara and I. Pastan. Small Chimeric Toxins Containing Only Transforming Growth Factor .alpha. and Domain III of Pseudomonas Exotoxin with Good Antitumor Activity in Mice. Cancer Res 1994:54:5154-5159). A modified form with an added OmpA signal sequence and extended flexible linker with enhanced activity functionally expressed, secreted, surface displayed and/or released is shown in FIG. 1B. The construct contains an OmpA secretion signal, TGF.alpha. targeting domain, a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA domain III followed by, optionally KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023 is used. The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1286 is:

TABLE-US-00003 SEQ ID NO.: 003 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTactggcgcggagttcctc ggcgacggcggcgacgtcagcttcagcacccgcggcacgcagaactggac ggtggagcggctgctccaggcgcaccgccaactggaggagcgcggctatg tgttcgtcggctaccacggcaccttcctcgaagcggcgcaaagcatcgtc ttcggcggggtgcgcgcgcgcagccaggacctcgacgcgatctggcgcgg tttctatatcgccggcgatccggcgctggcctacggctacgcccaggacc aggaacccgacgcacgcggccggatccgcaacggtgccctgctgcgggtc tatgtgccgcgctcgagcctgccgggcttctaccgcaccagcctgaccct ggccgcgccggaggcggcgggcgaggtcgaacggctgatcggccatccgc tgccgctgcgcctggacgccatcaccggccccgaggaggaaggcgggcgc ctggagaccattctcggctggccgctggccgagcgcaccgtggtgattcc ctcggcgatccccaccgacccgcgcaacgtcggcggcgacctcgacccgt ccagcatccccgacaaggaacaggcgatcagcgccctgccggactacgcc agccagcccggcaaaccgccgaaagatgaactgtaaCTCGAGTagatctg tactCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATG AGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTC TGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGAC CCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGG GTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAG GCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAA CGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGA AGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGG CATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCT ACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGA GTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCA TTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGA TGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCA ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACA GTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGC CAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAG CTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAG CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGA CCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATC TGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAG ATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGC GTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTC TGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTG GTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGG CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTAC CGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGA AGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC AGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCC CCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGC TCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG AAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCA CACCGCATATG

The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1286 together with the colicin E3 lysis protein from 1321 to 1464 is:

TABLE-US-00004 SEQ ID NO.: 004 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTctgacctgcccggtcgcc gccggtgaatgcgcgggcccggcggacagcggcgacgccctgctggagcg caactatcccactggcgcggagttcctcggcgacggcggcgacgtcagct tcagcacccgcggcacgcagaactggacggtggagcggctgctccaggcg caccgccaactggaggagcgcggctatgtgttcgtcggctaccacggcac cttcctcgaagcggcgcaaagcatcgtcttcggcggggtgcgcgcgcgca gccaggacctcgacgcgatctggcgcggtttctatatcgccggcgatccg gcgctggcctacggctacgcccaggaccaggaacccgacgcacgcggccg gatccgcaacggtgccctgctgcgggtctatgtgccgcgctcgagcctgc cgggcttctaccgcaccagcctgaccctggccgcgccggaggcggcgggc gaggtcgaacggctgatcggccatccgctgccgctgcgcctggacgccat caccggccccgaggaggaaggcgggcgcctggagaccattctcggctggc cgctggccgagcgcaccgtggtgattccctcggcgatccccaccgacccg cgcaacgtcggcggcgacctcgacccgtccagcatccccgacaaggaaca ggcgatcagcgccctgccggactacgccagccagcccggcaaaccgccga aagatgaactgtaaCTCGAGTagatctgtactCTAGAAAGGAGTCGTTAT GAAAAAAATAACAGGGATTATTTTATTGCTTCTTGCAGTCATTATTCTGT CTGCATGTCAGGCAAACTATATCCGGGATGTTCAGGGCGGGACCGTATCT CCGTCATCAACAGCTGAAGTGACCGGATTAGCAACGCAGTAACTGCAGGC ATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATAC AGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGC GGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAA ACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGA ACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATC CGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGG GCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCAT CCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAA TGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGT GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT ATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAAC TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC CGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT TACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 4

Modified TGF.alpha.-PE38 (Kreitman, R. J., Siegall, C. B., Chaudhary, V. K, Fitzgerald, D. J., and Pastan. I. Properties of Chimeric Toxins with Two Recognition Domains: Interleukin 6 and Transforming Growth Factor .alpha. at Different Locations in Pseudomonas Exotoxin. Bioconjugate Chem. 1992, 3, 63-68.)

The original form of this product represents a deletion of amino acids 365-380 of ToxA domain I. The modified form is a chimera containing an OmpA secretion signal, TGF.alpha. targeting domain, a GGGGS(.times.3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (.DELTA.365-380 amino acids) domain III followed by, optionally KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023 is used. Optionally, the colicin E3 lysis protein with a separate ribosomal binding site may be added. The modified form with an added extended flexible linker with enhanced activity functionally expressed, secreted, surface displayed and/or released is shown in FIG. 1C. The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1664 is:

TABLE-US-00005 SEQ ID NO.: 005 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCggggtggcagcctggccgcgctg accgcgcaccaggcttgccacctgccgctggagactttcacccgtcatcg ccagccgcgcggctgggaacaactggagcagtgcggctatccggtgcagc ggctggtcgccctctacctggcggcgcggctgtcgtggaaccaggtcgac caggtgatccgcaacgccctggccagccccggcagcggcggcgacctggg cgaagcgatccgcgagcagccggagcaggcccgtctggccctgaccctgg ccgccgccgagagcgagcgcttcgtccggcagggcaccggcaacgacgag gccggcgcggccaacgccccggcggacagcggcgacgccctgctggagcg caactatcccactggcgcggagttcctcggcgacggcggcgacgtcagct tcagcacccgcggcacgcagaactggacggtggagcggctgctccaggcg caccgccaactggaggagcgcggctatgtgttcgtcggctaccacggcac cttcctcgaagcggcgcaaagcatcgtcttcggcggggtgcgcgcgcgca gccaggacctcgacgcgatctggcgcggtttctatatcgccggcgatccg gcgctggcctacggctacgcccaggaccaggaacccgacgcacgcggccg gatccgcaacggtgccctgctgcgggtctatgtgccgcgctcgagcctgc cgggcttctaccgcaccagcctgaccctggccgcgccggaggcggcgggc gaggtcgaacggctgatcggccatccgctgccgctgcgcctggacgccat caccggccccgaggaggaaggcgggcgcctggagaccattctcggctggc cgctggccgagcgcaccgtggtgattccctcggcgatccccaccgacccg cgcaacgtcggcggcgacctcgacccgtccagcatccccgacaaggaaca ggcgatcagcgccctgccggactacgccagccagcccggcaaaccgccga aagatgaactgtaaCTCGAGTagatctgtactCTAGAGTCGACCTGCAGG CATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATA CAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGG CGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGA AACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGG AACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCT TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT CCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCG GGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCA TCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTT TCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG TGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG TTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGC TATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCAC AGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAA CTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC AGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCC CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTC TGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTG AGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGAC TTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT TGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTA TTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAG CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTT TCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a start codon at 351 and a stop codon at 1664 together with the colicin E3 lysis protein from 1699 to 1842 is:

TABLE-US-00006 SEQ ID NO.: 006 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGAATTCACC ATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGC TACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATTGCCCGGATA GCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCTGGTGCAGGAA GATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGCGCTCGCTGCGA ACATGCGGATCTGCTGGCccATCAACTAGTGGGCGGTGGTGGCAGTGGTG GCGGCGGCTCTGGTGGTGGCGGGTCCCggggtggcagcctggccgcgctg accgcgcaccaggcttgccacctgccgctggagactttcacccgtcatcg ccagccgcgcggctgggaacaactggagcagtgcggctatccggtgcagc ggctggtcgccctctacctggcggcgcggctgtcgtggaaccaggtcgac caggtgatccgcaacgccctggccagccccggcagcggcggcgacctggg cgaagcgatccgcgagcagccggagcaggcccgtctggccctgaccctgg ccgccgccgagagcgagcgcttcgtccggcagggcaccggcaacgacgag gccggcgcggccaacgccccggcggacagcggcgacgccctgctggagcg caactatcccactggcgcggagttcctcggcgacggcggcgacgtcagct tcagcacccgcggcacgcagaactggacggtggagcggctgctccaggcg caccgccaactggaggagcgcggctatgtgttcgtcggctaccacggcac cttcctcgaagcggcgcaaagcatcgtcttcggcggggtgcgcgcgcgca gccaggacctcgacgcgatctggcgcggtttctatatcgccggcgatccg gcgctggcctacggctacgcccaggaccaggaacccgacgcacgcggccg gatccgcaacggtgccctgctgcgggtctatgtgccgcgctcgagcctgc cgggcttctaccgcaccagcctgaccctggccgcgccggaggcggcgggc gaggtcgaacggctgatcggccatccgctgccgctgcgcctggacgccat caccggccccgaggaggaaggcgggcgcctggagaccattctcggctggc cgctggccgagcgcaccgtggtgattccctcggcgatccccaccgacccg cgcaacgtcggcggcgacctcgacccgtccagcatccccgacaaggaaca ggcgatcagcgccctgccggactacgccagccagcccggcaaaccgccga aagatgaactgtaaCTCGAGTagatctgtactCTAGAAAGGAGTCGTTAT GAAAAAAATAACAGGGATTATTTTATTGCTTCTTGCAGTCATTATTCTGT CTGCATGTCAGGCAAACTATATCCGGGATGTTCAGGGCGGGACCGTATCT CCGTCATCAACAGCTGAAGTGACCGGATTAGCAACGCAGTAACTGCAGGC ATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATAC AGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGC GGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAA ACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGA ACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATC CGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGG GCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCAT CCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAA TGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGT GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT ATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAAC TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC CGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT TACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 5

An artificial typhoid toxin operon lacking sty and ttsA.

An artificial, inducible typhoid toxin operon lacking sty and ttsA as shown in FIG. 2B, is generated by multiple PCR and restriction endonuclease cloning steps known to those skilled in the art. The operon is capable of being induced with an inducing agent, in the present instance, arabinose. As compared with FIG. 1B, the artificial operon lacks the sty and ttsA genes. The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a cldtB start codon at 351 and a stop codon at 1163, pltB start at 1197 and stop at 1610 and pltA 1627-2355:

TABLE-US-00007 SEQ ID NO.: 007 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCC ATGGTCAAAAAACCTGTTTTTTTCCTTCTGACCATGATCATCTGCAGCTA TATTTCTTTTGCCTGCGCTAATATCAGTGACTACAAAGTTATGACCTGGA ATCTTCAGGGCTCTTCAGCATCTACAGAAAGTAAATGGAATGTCAATGTC AGACAGCTTTTAAGCGGTACTGCCGGTGTGGATATTCTTATGGTACAGGA GGCCGGTGCTGTTCCCACCTCAGCGGTTCCTACCGGACGACATATTCAGC CTTTTGGAGTGGGTATTCCCATTGATGAATACACCTGGAATCTCGGAACC ACCAGCCGTCAGGATATAAGATATATCTACTACTCGGCTATTGATGTTGG AGCACGCCGTGTTAATCTGGCAATAGTTTCCAGACAAAGAGCGGATAATG TTTATGTCTTGCGTCCGACAACTGTCGCATCTCGCCCCGTCATTGGCATC GGACTGGGTAATGATGTTTTTCTGACAGCGCACGCACTGGCTAGTGGAGG TCCGGATGCTGCAGCTATTGTCAGGGTTACCATTAATTTTTTTAGACAAC CTCAGATGCGGCATTTATCCTGGTTTCTTGCCGGGGATTTTAATCGCAGC CCAGACAGACTTGAAAATGACCTGATGACTGAGCATCTGGAACGAGTTGT AGCCGTACTCGCACCTACAGAACCCACGCAAATTAGCGGTGGTATTTTAG ATTATGGGGTCATTGTCGATCGAGCACCTTATTCACAAAGGGTCGAAGCA TTACGTAATCCACAACTCGCTTCTGATCATTATCCCGTAGCCTTTTTGGC ACGAAGCTGTTAAtgaTCTAGATAAGTCGACgtacAGGAgagtagtATGT ATATAAATAAGTTTGTGCCTGTTTATACATTATTAATTCTCATTTATTCT TTTAATGCCAGCGCTGAGTGGACAGGAGATAATACGAACGCCTATTACTC AGACGAAGTTATCAGTGAATTACATGTTGGTCAGATAGATACTAGTCCTT ATTTTTGCATAAAAACGGTTAAAGCTAACGGTAGTGGTACACCAGTTGTT GCATGTGCGGTATCAAAGCAGAGCATATGGGCGCCCTCCTTTAAAGAACT TCTTGATCAGGCAAGATATTTTTACAGTACAGGGCAATCCGTAAGGATTC ATGTTCAAAAAAATATCTGGACCTATCCGCTTTTTGTTAATACCTTTTCA GCAAATGCTCTTGTGGGACTATCATCGTGCAGTGCGACACAATGCTTTGG ACCCAAGTAAgaggggagaagaaataATGAAAAAGTTAATATTCTTAACC TTATCTATAGTTAGCTTTAATAACTATGCTGTAGATTTTGTGTATCGTGT GGACTCAACCCCGCCGGACGTTATTTTTCGCGATGGGTTTTCACTACTTG GGTATAACCGTAACTTTCAGCAATTTATTAGTGGAAGGTCATGTAGTGGT GGAAGTAGTGACAGTCGCTATATTGCAACAACCTCAAGTGTTAATCAAAC ATATGCTATAGCCAGAGCGTACTATTCTCGCTCAACATTCAAAGGTAATT TATACAGATATCAGATTCGTGCAGATAATAATTTCTACAGCTTGCTCCCA TCCATCACCTATCTGGAAACGCAAGGTGGTCACTTTAATGCTTATGAAAA AACGATGATGCGATTGCAAAGAGAGTATGTTTCCACATTATCTATTTTAC CCGAGAATATTCAAAAGGCCGTGGCGCTAGTTTATGATAGCGCAACCGGT CTGGTAAAGGATGGTGTAAGCACAATGAATGCCAGTTATTTAGGTTTAAG CACTACGTCTAATCCTGGTGTGATACCTTTTCTTCCGGAACCGCAGACGT ATACCCAACAACGAATTGATGCATTCGGCCCATTAATAAGTTCATGCTTT TCAATAGGTAGCGTATGTCAGTCACATCGAGGGCAAAGAGCTGACGTATA CAACATGTCTTTTTATGATGCAAGACCTGTAATAGAACTTATACTTTCTA AATAAatgaaacttacctatgttgccGCATGCAAGCTAGCTTGGCTGTTT TGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCA GAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTC CCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGG TAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATA AAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT GTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGA ACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAA ACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTT TGTTTCGCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATAT GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAA AAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTT TTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAA AGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAAC TGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT TTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC CCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT GCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGC TGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGG AGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGC CTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATAC TTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATAC CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG ACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCT TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCG AGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGC GGTATTTCACACCGCATATG

Example 6

An artificial typhoid toxin operon lacking sty and ttsA with introduced cloning sites FLAG epitope and GGGGS(.times.3) SEQ ID NO.: 008 linker at the C-terminus of cldtB.

An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced cloning sites FLAG epitope and GGGGS(.times.3) SEQ ID NO.: 008 linker at the C-terminus as shown in FIG. 2D, is generated by multiple overlapping PCR and restriction endonuclease cloning steps and synthetic biology known to those skilled in the art. The operon is capable of being induced with an inducing agent, in the present instance, arabinose. As compared with FIG. 2B, the artificial operon now contains an artificial, in-frame restriction endonuclease linker containing SexAl, HindIII and Xbal the FLAG epitope and a GGGGS(.times.3) SEQ ID NO.: 008 flexible linker. The complete sequence of the arabinose inducible plasmid capable of expressing the construct with a cldtB start codon at 351 through codon at 1160 (without a stop), FLAG from 1170-1193, and GGGGS(.times.3) SEQ ID NO.: 008 from 1194-1238, pltB start at 1278 and stop at 1691 and pltA 1708-2436:

TABLE-US-00008 SEQ ID NO.: 009 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCC atgGTCaaaaaacctgtttttttccttctgaccatgatcatctgcagcta tatttcttttgcctgcgctaatatcagtgactacaaagttatgacctgga atcttcagggctcttcagcatctacagaaagtaaatggaatgtcaatgtc agacagcttttaagcggtactgccggtgtggatattcttatggtacagga ggccggtgctgttcccacctcagcggttcctaccggacgacatattcagc cttttggagtgggtattcccattgatgaatacacctggaatctcggaacc accagccgtcaggatataagatatatctactactcggctattgatgttgg agcacgccgtgttaatctggcaatagtttccagacaaagagcggataatg tttatgtcttgcgtccgacaactgtcgcatctcgccccgtcattggcatc ggactgggtaatgatgtttttctgacagcgcacgcactggctagtggagg tccggatgctgcagctattgtcagggttaccattaatttttttagacaac ctcagatgcggcatttatcctggtttcttgccggggattttaatcgcagc ccagacagacttgaaaatgacctgatgactgagcatctggaacgagttgt agccgtactcgcacctacagaacccacgcaaattagcggtggtattttag attatggggtcattgtcgatcgagcaccttattcacaaagggtcgaagca ttacgtaatccacaactcgcttctgatcattatcccgtagcctttttggc acgaagctgtaTAccaggtgattataaagatgacgatgacaaaggcggtg gcggtagcggcggtggcggttctggcggtggcggtagtaagcttGGGtct agaTAGgtcgacgtacAGGAgagtagtATGTATATAAATAAGTTTGTGCC TGTTTATACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAGT GGACAGGAGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGAA TTACATGTTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGGT TAAAGCTAACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAGC AGAGCATATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATAT TTTTACAGTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCTG GACCTATCCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGAC TATCATCGTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgaggggaga agaaataATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCTTTA ATAACTATGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCCGGAC GTTATTTTTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAACTTTCA GCAATTTATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACAGTCGCT ATATTGCAACAACCTCAAGTGTTAATCAAACATATGCTATAGCCAGAGCG TACTATTCTCGCTCAACATTCAAAGGTAATTTATACAGATATCAGATTCG TGCAGATAATAATTTCTACAGCTTGCTCCCATCCATCACCTATCTGGAAA CGCAAGGTGGTCACTTTAATGCTTATGAAAAAACGATGATGCGATTGCAA AGAGAGTATGTTTCCACATTATCTATTTTACCCGAGAATATTCAAAAGGC CGTGGCGCTAGTTTATGATAGCGCAACCGGTCTGGTAAAGGATGGTGTAA GCACAATGAATGCCAGTTATTTAGGTTTAAGCACTACGTCTAATCCTGGT GTGATACCTTTTCTTCCGGAACCGCAGACGTATACCCAACAACGAATTGA TGCATTCGGCCCATTAATAAGTTCATGCTTTTCAATAGGTAGCGTATGTC AGTCACATCGAGGGCAAAGAGCTGACGTATACAACATGTCTTTTTATGAT GCAAGACCTGTAATAGAACTTATACTTTCTAAATAAatgaaacttaccta tgttgccGCATGCAAGCTAGCTTGGCTGTTTTGGCGGATGAGAGAAGATT TTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACA GAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGA ACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCAT GCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGA AAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTG AGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCC CGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTA AGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTT TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAA TAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTAT TCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAA GAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT TATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACAT GGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAG CCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACA ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA ACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGT GAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG AACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGG TAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACT TCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGC CGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCA CTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGG TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGAT ACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG GCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC GCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGC CGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCT GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAT G

Example 7

Apoptin C-terminal fusions with typhoid toxin cldtB.

An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced FLAG epitope, GGGGS(.times.3) SEQ ID NO.: 008 linker and HindIII and Xbal is used to insert apoptin, TAT-apoptin, and apoptin fragments as shown in FIG. 2E as generated by PCR and restriction endonuclease-based cloning methods and synthetic biology known to those skilled in the art. Fragments consisting of either 1) apoptin 1-121, 2) apoptin 33-121, 3) apoptin 82-121, 4) apoptin 97-121, 5) apoptin 106-121, 6) apoptin 111-121 or 7) apoptin 1-31 linked ot 83-121. By way of example complete sequence of the arabinose inducible plasmid capable of expressing the TAT-apoptin construct as a cldtB fusion, with TAT-apoptin inserted in-frame within the HindIII and Xbal sites introduced into typhoid toxin cldtB, with the TAT-apoptin coding sequence from 1245-1646:

TABLE-US-00009 SEQ ID NO.: 010 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCC atgGTCaaaaaacctgtttttttccttctgaccatgatcatctgcagcta tatttcttttgcctgcgctaatatcagtgactacaaagttatgacctgga atcttcagggctcttcagcatctacagaaagtaaatggaatgtcaatgtc agacagcttttaagcggtactgccggtgtggatattcttatggtacagga ggccggtgctgttcccacctcagcggttcctaccggacgacatattcagc cttttggagtgggtattcccattgatgaatacacctggaatctcggaacc accagccgtcaggatataagatatatctactactcggctattgatgttgg agcacgccgtgttaatctggcaatagtttccagacaaagagcggataatg tttatgtcttgcgtccgacaactgtcgcatctcgccccgtcattggcatc ggactgggtaatgatgtttttctgacagcgcacgcactggctagtggagg tccggatgctgcagctattgtcagggttaccattaatttttttagacaac ctcagatgcggcatttatcctggtttcttgccggggattttaatcgcagc ccagacagacttgaaaatgacctgatgactgagcatctggaacgagttgt agccgtactcgcacctacagaacccacgcaaattagcggtggtattttag attatggggtcattgtcgatcgagcaccttattcacaaagggtcgaagca ttacgtaatccacaactcgcttctgatcattatcccgtagcctttttggc acgaagctgtaTAccaggtgattataaagatgacgatgacaaaggcggtg gcggtagcggcggtggcggttctggcggtggcggtagtaagcttATGGCA TACGGTCGCAAGAAACGTCGTCAACGCCGCCGCATGAACGCCCTGCAAGA AGACACGCCGCCGGGTCCGAGCACGGTTTTTCGTCCGCCGACCAGCTCTC GCCCGCTGGAAACGCCGCATTGCCGTGAAATTCGCATCGGCATTGCAGGT ATCACCATTACGCTGTCTCTGTGCGGCTGTGCAAACGCACGTGCACCGAC CCTGCGCTCCGCTACGGCGGATAACAGTGAATCCACCGGTTTTAAAAATG TGCCGGATCTGCGTACGGACCAGCCGAAGCCGCCGTCAAAAAAGCGTTCG TGTGACCCGAGCGAATATCGCGTTAGCGAACTGAAAGAATCTCTGATTAC CACGACCCCGTCCCGTCCGCGCACCGCaaaACGCCGCATCCGTCTGTCTA GATAGgtcgacgtacAGGAgagtagtATGTATATAAATAAGTTTGTGCCT GTTTATACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAGTG GACAGGAGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGAAT TACATGTTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGGTT AAAGCTAACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAGCA GAGCATATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATATT TTTACAGTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCTGG ACCTATCCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGACT ATCATCGTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgaggggagaa gaaataATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCTTTAA TAACTATGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCCGGACG TTATTTTTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAACTTTCAG CAATTTATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACAGTCGCTA TATTGCAACAACCTCAAGTGTTAATCAAACATATGCTATAGCCAGAGCGT ACTATTCTCGCTCAACATTCAAAGGTAATTTATACAGATATCAGATTCGT GCAGATAATAATTTCTACAGCTTGCTCCCATCCATCACCTATCTGGAAAC GCAAGGTGGTCACTTTAATGCTTATGAAAAAACGATGATGCGATTGCAAA GAGAGTATGTTTCCACATTATCTATTTTACCCGAGAATATTCAAAAGGCC GTGGCGCTAGTTTATGATAGCGCAACCGGTCTGGTAAAGGATGGTGTAAG CACAATGAATGCCAGTTATTTAGGTTTAAGCACTACGTCTAATCCTGGTG TGATACCTTTTCTTCCGGAACCGCAGACGTATACCCAACAACGAATTGAT GCATTCGGCCCATTAATAAGTTCATGCTTTTCAATAGGTAGCGTATGTCA GTCACATCGAGGGCAAAGAGCTGACGTATACAACATGTCTTTTTATGATG CAAGACCTGTAATAGAACTTATACTTTCTAAATAAatgaaacttacctat gttgccGCATGCAAGCTAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTT TCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAG AATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAA CTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATG CGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAA AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGA GTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCC GGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAA GCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTT TTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAAT AACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCC TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAG ATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAG AGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTAC TCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATG GGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGC CATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAA CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTG AGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCC TCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATC TGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG CGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTT ACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGT TCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTG ATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 8

N-terminal fusions with typhoid toxin cldtB.

An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced FLAG epitope, GGGGS(.times.3) SEQ ID NO.: 008 linker and HindIII and Xbal is used to insert functional peptides in frame (e.g., apoptin, TAT-apoptin, and apoptin fragments) as shown in FIG. 3B as generated by PCR and restriction endonuclease-based cloning methods and synthetic biology known to those skilled in the art. For example, two separate PCR primer sets that generate N-terminal restriction sites (HindIII (H3) Xbal and Clal) for cloning typhoid/paratyphoid cldtB are

TABLE-US-00010 paratyphi A NcoI_F1: SEQ ID NO.: 011 5'-TACGCCATGGTCAAAAAACCTGTTTTTTTCCTTCTGACC-3' and HindIII_Xbal_Clal-R1: SEQ ID NO.: 012 5'-CGATCCCTCTAGACCCAAGCTTGTCACTGATATTAGCGCAGGC-3' (set 1) and HindIII_Xbal_Clal-F1: SEQ ID NO.: 13 5'-GCTTGGGTCTAGAGGGATCGATTACAAAGTTATGACCTGGAATCTTC AG-3' and cldtB_stop_Xbal_Sall-R1: SEQ ID NO.: 14 5'- GATCGTCGACTTATCTAGATCATTAACAGCTTCGTGCCA-3' (set 2).

The two sets are used for separate PCRs (for example, using the DNA of FIG. 2B as a template) followed by overlapping PCR and cloning into the Ncol and Xbal restriction endonuclease sites (13-350=arabinose promoter; 351-1187=cldtB; 1210-1213=RBS pltB; 1221-1634=pltB; 1651-2379=pltA).

TABLE-US-00011 SEQ ID NO.: 015 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCC ATGGTCAAAAAACCTGTTTTTTTCCTTCTGACCATGATCATCTGCAGCTA TATTTCTTTTGCCTGCGCTAATATCAGTGACaagcttgggtctagaggga tcgatTACAAAGTTATGACCTGGAATCTTCAGGGCTCTTCAGCATCTACA GAAAGTAAATGGAATGTCAATGTCAGACAGCTTTTAAGCGGTACTGCCGG TGTGGATATTCTTATGGTACAGGAGGCCGGTGCTGTTCCCACCTCAGCGG TTCCTACCGGACGACATATTCAGCCTTTTGGAGTGGGTATTCCCATTGAT GAATACACCTGGAATCTCGGAACCACCAGCCGTCAGGATATAAGATATAT CTACTACTCGGCTATTGATGTTGGAGCACGCCGTGTTAATCTGGCAATAG TTTCCAGACAAAGAGCGGATAATGTTTATGTCTTGCGTCCGACAACTGTC GCATCTCGCCCCGTCATTGGCATCGGACTGGGTAATGATGTTTTTCTGAC AGCGCACGCACTGGCTAGTGGAGGTCCGGATGCTGCAGCTATTGTCAGGG TTACCATTAATTTTTTTAGACAACCTCAGATGCGGCATTTATCCTGGTTT CTTGCCGGGGATTTTAATCGCAGCCCAGACAGACTTGAAAATGACCTGAT GACTGAGCATCTGGAACGAGTTGTAGCCGTACTCGCACCTACAGAACCCA CGCAAATTAGCGGTGGTATTTTAGATTATGGGGTCATTGTCGATCGAGCA CCTTATTCACAAAGGGTCGAAGCATTACGTAATCCACAACTCGCTTCTGA TCATTATCCCGTAGCCTTTTTGGCACGAAGCTGTTAAtgaTCTAGATAAG TCGACgtacAGGAgagtagtATGTATATAAATAAGTTTGTGCCTGTTTAT ACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAGTGGACAGG AGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGAATTACATG TTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGGTTAAAGCT AACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAGCAGAGCAT ATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATATTTTTACA GTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCTGGACCTAT CCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGACTATCATC GTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgaggggagaagaaata ATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCTTTAATAACTA TGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCCGGACGTTATTT TTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAACTTTCAGCAATTT ATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACAGTCGCTATATTGC AACAACCTCAAGTGTTAATCAAACATATGCTATAGCCAGAGCGTACTATT CTCGCTCAACATTCAAAGGTAATTTATACAGATATCAGATTCGTGCAGAT AATAATTTCTACAGCTTGCTCCCATCCATCACCTATCTGGAAACGCAAGG TGGTCACTTTAATGCTTATGAAAAAACGATGATGCGATTGCAAAGAGAGT ATGTTTCCACATTATCTATTTTACCCGAGAATATTCAAAAGGCCGTGGCG CTAGTTTATGATAGCGCAACCGGTCTGGTAAAGGATGGTGTAAGCACAAT GAATGCCAGTTATTTAGGTTTAAGCACTACGTCTAATCCTGGTGTGATAC CTTTTCTTCCGGAACCGCAGACGTATACCCAACAACGAATTGATGCATTC GGCCCATTAATAAGTTCATGCTTTTCAATAGGTAGCGTATGTCAGTCACA TCGAGGGCAAAGAGCTGACGTATACAACATGTCTTTTTATGATGCAAGAC CTGTAATAGAACTTATACTTTCTAAATAAatgaaacttacctatgttgcc GCATGCAAGCTAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCC TGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTG CCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGA AGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAG TAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGA CAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGG TGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAA GGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTT ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTT TGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGT TCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAAC TCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAG TGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC AAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGC GCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTG GGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGT ATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTT TAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAA AATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA TACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGAC GATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACA GCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACA GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT CCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGA CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGG TATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 9

N-terminal fusions with typhoid toxin cldtB containing a 3XFLAG epitope and a flexible linker.

The artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced HindIII (H3) Xbal and Clal of Example 8 is used to insert an in-frame 3XFLAG epitope (for immune identification and/or purification) and a flexible linker (GGGGS(.times.3), SEQ ID NO.: 008 as shown in FIG. 3C as generated by PCR and restriction endonuclease-based cloning methods and synthetic biology known to those skilled in the art (13-350=arabinose promoter; 351-1295=cldtB; 1318-1321=RBS pltB; 1329-1742=pltB; 1759-2487=pltA).

TABLE-US-00012 SEQ ID NO.: 016 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgt cactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgct tattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgc gtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttg cacggcgtcacactttgctatgccatagcatttttatccataagattagc ggatcctacctgacgctttttatcgcaactctctactgtttctccatacc cgtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCC ATGGTCAAAAAACCTGTTTTTTTCCTTCTGACCATGATCATCTGCAGCTA TATTTCTTTTGCCTGCGCTAATATCAGTGACaagcttgggtctagagact acaaagaccatgacggtgattataaagatcatgatatcgactacaaagat gacgacgataaaGGCGGTGGCGGATCCGGCGGTGGCGGTTCTGGCGGTGG CGGTAGTatcgatTACAAAGTTATGACCTGGAATCTTCAGGGCTCTTCAG CATCTACAGAAAGTAAATGGAATGTCAATGTCAGACAGCTTTTAAGCGGT ACTGCCGGTGTGGATATTCTTATGGTACAGGAGGCCGGTGCTGTTCCCAC CTCAGCGGTTCCTACCGGACGACATATTCAGCCTTTTGGAGTGGGTATTC CCATTGATGAATACACCTGGAATCTCGGAACCACCAGCCGTCAGGATATA AGATATATCTACTACTCGGCTATTGATGTTGGAGCACGCCGTGTTAATCT GGCAATAGTTTCCAGACAAAGAGCGGATAATGTTTATGTCTTGCGTCCGA CAACTGTCGCATCTCGCCCCGTCATTGGCATCGGACTGGGTAATGATGTT TTTCTGACAGCGCACGCACTGGCTAGTGGAGGTCCGGATGCTGCAGCTAT TGTCAGGGTTACCATTAATTTTTTTAGACAACCTCAGATGCGGCATTTAT CCTGGTTTCTTGCCGGGGATTTTAATCGCAGCCCAGACAGACTTGAAAAT GACCTGATGACTGAGCATCTGGAACGAGTTGTAGCCGTACTCGCACCTAC AGAACCCACGCAAATTAGCGGTGGTATTTTAGATTATGGGGTCATTGTCG ATCGAGCACCTTATTCACAAAGGGTCGAAGCATTACGTAATCCACAACTC GCTTCTGATCATTATCCCGTAGCCTTTTTGGCACGAAGCTGTTAAtgaTC TAGATAAGTCGACgtacAGGAgagtagtATGTATATAAATAAGTTTGTGC CTGTTTATACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAG TGGACAGGAGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGA ATTACATGTTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGG TTAAAGCTAACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAG CAGAGCATATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATA TTTTTACAGTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCT GGACCTATCCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGA CTATCATCGTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgaggggag aagaaataATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCTTT AATAACTATGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCCGGA CGTTATTTTTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAACTTTC AGCAATTTATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACAGTCGC TATATTGCAACAACCTCAAGTGTTAATCAAACATATGCTATAGCCAGAGC GTACTATTCTCGCTCAACATTCAAAGGTAATTTATACAGATATCAGATTC GTGCAGATAATAATTTCTACAGCTTGCTCCCATCCATCACCTATCTGGAA ACGCAAGGTGGTCACTTTAATGCTTATGAAAAAACGATGATGCGATTGCA AAGAGAGTATGTTTCCACATTATCTATTTTACCCGAGAATATTCAAAAGG CCGTGGCGCTAGTTTATGATAGCGCAACCGGTCTGGTAAAGGATGGTGTA AGCACAATGAATGCCAGTTATTTAGGTTTAAGCACTACGTCTAATCCTGG TGTGATACCTTTTCTTCCGGAACCGCAGACGTATACCCAACAACGAATTG ATGCATTCGGCCCATTAATAAGTTCATGCTTTTCAATAGGTAGCGTATGT CAGTCACATCGAGGGCAAAGAGCTGACGTATACAACATGTCTTTTTATGA TGCAAGACCTGTAATAGAACTTATACTTTCTAAATAAatgaaacttacct atgttgccGCATGCAAGCTAGCTTGGCTGTTTTGGCGGATGAGAGAAGAT TTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAAC AGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCG AACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCA TGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCG AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCT GAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGC CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATT AAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCT TTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACA ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT CCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTA AGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT TTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACT TCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA GCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAAC AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTG CGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGAT GAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTG GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTC ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAG AGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACC ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTG TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGA CTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGA TACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAA GGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCG GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCT TTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCT GTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAG CCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATA TG

Functional in-frame peptide fragments such as those consisting of either 1) apoptin 1-121, 2) apoptin 33-121, 3) apoptin 82-121, 4) apoptin 97-121, 5) apoptin 106-121, 6) apoptin 111-121 or 7) apoptin 1-31 linked to 83-121 are further constructed by PCR and restriction endonuclease-based cloning methods and synthetic biology known to those skilled in the art as shown in FIG. 3D.

Example 10

Cytotoxicity of the cloned S. paratyphi cytolethal distending toxin wild type and reorganized operon with an inserted polylinker, and apoptin fusions. Cytotoxicity of Salmonella paratyphi A Cytolethal distending toxin toward MDA-MB-468 breast cancer cells is conducted using methods known to those skilled in the art and is measured by the MTT assay where absorbance at 570 nm indicates the relative number of live cells. (FIGS. 8A and 8B). By comparing no treatment control and a bacterial culture supernatant carrying an empty vector plasmid into a 100 .mu.L tissue culture well control with a the reorganized polycistronic S. paratyphi A cytolethal distending operon containing a polylinker in cldtB C-terminus with dilutions of 1/2, 1/4, 1/8, and 1/16 and the reorganized polycistronic S. paratyphi A cytolethal distending operon without a polylinker in cldtB C-terminus, the ability of the CLDT is demonstrated, and the ability to secrete CLDT from the reorganized operon lacking sty and ttsA is demonstrated, as well as the ability to accommodate a polylinker.

Example 11

C-terminal fusions with typhoid toxin cldtB and a modified apoptin wherein the phosphorylation site T108 and the two adjacent threonines 106 and 107 are mutated to alanines does not alter cytotoxicity. The mutations are made by methods known to those skilled in the art. Modification of the phosphorylation sites abrogates the activity of apoptin. The resulting cytotoxicity test shows that there was no change in cytotoxicity, therefore, the cldtB:apoptin fusion does not acquire cell killing ability from the apoptin.

Example 12

A chimeric YebF:SFTI. The chimeric sunflower trypsin inhibitor:YebF fusion is made by methods known to those skilled in the art. The complete sequence of a tetracycline-inducible YebF:SFTI the start codon begins at 724 and the stop codon ends at 1158 is given by:

TABLE-US-00013 SEQ ID NO.: 017 ATATGGGGGCGGCCGCCTAAGACCCACTTTCACATTTAAGTTGTTTTTCT AATCCGCATATAATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACC TTGGTGTTCAAATAATTCGATAGCTTGTCGTAATAATGCCGGCATACTAT CAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCT TCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAA TGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTT CGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTT GCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATT ACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTAT GGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTA TTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCCAGTTTCTGGGC GAGTTTACGGGTTTTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTA ATGCGCTGTTAATCACTTTACTTTTATCTAATCTGGACATCATAAATTCC TAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTA TCAGTGATAGAGAAAAGTGAACCatggctaaaaaaagaggggcgttttta gggctgttgttggtttctgcctgcgcatcagttttcgctgccaataatga aaccagcaagtcggtcactttcccaaagtgtgaagatctggatgctgccg gaattgccgcgagcgtaaaacgtgattatcaacaaaatcgcgtggcgcgt tgggcagatgatcaaaaaattgtcggtcaggccgatcccgtggcttgggt cagtttgcaggacattcagggtaaagatgataaatggtcagtaccgctaa ccgtgcgtggtaaaagtgccgatattcattaccaggtcagcgtggactgc aaagcgggaatggcggaatatcagcggcgtCTCGAGGACGATGACGATAA GGGTACCCTGAAAGGCCGCTGCACCAAAAGCATTCCGCCGATTTGCTTTC CGGATTAGTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGC GGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAG CGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCAC CTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGT GTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAAC GAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG GTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGT TGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTG CCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCG TTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG CGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTA AAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTC CAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT GTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAA TGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCA TGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGC TTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAAC CGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCT ACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTAC TCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGAT AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTC AGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTA GATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCAC TGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTA ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCC GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTT CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTT ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGA AGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTA TTTCACACCG

Example 13

A chimeric OmpA:SFTI. The chimeric sunflower trypsin inhibitor:OmpA fusion is made by methods known to those skilled in the art. The complete sequence of a tetracycline inducible OmpA:SFTI fusion of which the coding sequence begins with an ATG start codon begins at 725 and the TAG stop codon ends at 1008 that can be inserted into an expression vector is given by:

TABLE-US-00014 SEQ ID NO.: 018 CATATGGGGGCGGCCGCCTAAGACCCACTTTCACATTTAAGTTGTTTTTC TAATCCGCATATAATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCAC CTTGGTGTTCAAATAATTCGATAGCTTGTCGTAATAATGCCGGCATACTA TCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATC TTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATA ATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTT TCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTT TGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTAT TACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTA TGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTT ATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCCAGTTTCTGGG CGAGTTTACGGGTTTTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCT AATGCGCTGTTAATCACTTTACTTTTATCTAATCTGGACATCATAAATTC CTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCT ATCAGTGATAGAGAAAAGTGAACCATGGCTAAAAAGACAGCTATCGCGAT TGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGCTCCGAAAG ATGGCCGCTGCACCAAAAGCATTCCGCCGATTTGCTTTCCGGATTAGTCT AGAGTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAA GATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAA AACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATG CCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCC CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAG TCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT CCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAAC GGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAA ATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAAC TCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGA GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGC CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCG GTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGC ACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGG GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTT ACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACA ACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAAT GAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGC AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTT CTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTA AGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATG GATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCA TTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCG TAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCC ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG AGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAG AAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGC GCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG

Example 14

A chimeric OmpA:SFTI RBS E3 Lysis. The chimeric sunflower trypsin inhibitor:OmpA fusion is made by methods known to those skilled in the art. The complete sequence of a tetracycline inducible OmpA:SFTI fusion of which the coding sequence begins with an ATG start codon that begins at 725 and the TAG stop codon ends at 1008 followed by a Shine Delgarno sequence (RBS) and coding sequence for the E3 Lysis sequence of which begins with an ATG start codon that begins at 865 and ends with a TAA stop codon at 1008 that can be inserted into an expression vector is given by:

TABLE-US-00015 SEQ ID NO.: 019 CATATGGGGGCGGCCGCCTAAGACCCACTTTCACATTTAAGTTGTTTTTC TAATCCGCATATAATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCAC CTTGGTGTTCAAATAATTCGATAGCTTGTCGTAATAATGCCGGCATACTA TCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATC TTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATA ATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTT TCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTT TGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTAT TACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTA TGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTT ATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCCAGTTTCTGGG CGAGTTTACGGGTTTTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCT AATGCGCTGTTAATCACTTTACTTTTATCTAATCTGGACATCATAAATTC CTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCT ATCAGTGATAGAGAAAAGTGAACCATGGCTAAAAAGACAGCTATCGCGAT TGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGCTCCGAAAG ATGGCCGCTGCACCAAAAGCATTCCGCCGATTTGCTTTCCGGATTAGTCT AGAAAGGAGTCGTTATGAAAAAAATAACAGGGATTATTTTATTGCTTCTT GCAGTCATTATTCTGTCTGCATGTCAGGCAAACTATATCCGGGATGTTCA GGGCGGGACCGTATCTCCGTCATCAACAGCTGAAGTGACCGGATTAGCAA CGCAGTAACTGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAG ATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAA ACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCC CATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGT CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC CTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACG GCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAA TTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACT CTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA CAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC TTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCA CTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGA GTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATG AAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCA ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCC GGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCAT TGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAA ACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGAC CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGT AATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGA AAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCAC GAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATT CTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGC AGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCG CCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG

While the invention is shown by way of various examples and explanations, it should be understood that this specification and the drawings are intended to encompass the various combinations, sub-combinations, and permutations of the various features disclosed, and not limited by the particular combinations and sequences presented by way of example.

SEQUENCE LISTINGS

1

2313789DNAArtificial SequenceModified TGF-alpha-GIII-KDEL 1gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtctgacctg cccggtcgcc gccggtgaat 660gcgcgggccc ggcggacagc ggcgacgccc tgctggagcg caactatccc actggcgcgg 720agttcctcgg cgacggcggc gacgtcagct tcagcacccg cggcacgcag aactggacgg 780tggagcggct gctccaggcg caccgccaac tggaggagcg cggctatgtg ttcgtcggct 840accacggcac cttcctcgaa gcggcgcaaa gcatcgtctt cggcggggtg cgcgcgcgca 900gccaggacct cgacgcgatc tggcgcggtt tctatatcgc cggcgatccg gcgctggcct 960acggctacgc ccaggaccag gaacccgacg cacgcggccg gatccgcaac ggtgccctgc 1020tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta ccgcaccagc ctgaccctgg 1080ccgcgccgga ggcggcgggc gaggtcgaac ggctgatcgg ccatccgctg ccgctgcgcc 1140tggacgccat caccggcccc gaggaggaag gcgggcgcct ggagaccatt ctcggctggc 1200cgctggccga gcgcaccgtg gtgattccct cggcgatccc caccgacccg cgcaacgtcg 1260gcggcgacct cgacccgtcc agcatccccg acaaggaaca ggcgatcagc gccctgccgg 1320actacgccag ccagcccggc aaaccgccga aagatgaact gtaactcgag tagatctgta 1380ctctagagtc gacctgcagg catgcaagct tggctgtttt ggcggatgag agaagatttt 1440cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg 1500cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag 1560cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa 1620aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 1680ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg 1740gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca 1800tcctgacgga tggccttttt gcgtttctac aaactctttt tgtttatttt tctaaataca 1860ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 1920aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 1980ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 2040gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 2100ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 2160ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 2220gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 2280aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 2340gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 2400aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 2460caccacgatg cctacagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 2520tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 2580acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2640gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 2700agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 2760gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 2820ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 2880taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2940agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3000aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3060ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 3120gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3180aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3240aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3300gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3360aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3420aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3480cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3540cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3600tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3660tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3720ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 3780ccgcatatg 378923938DNAArtificial SequenceModified TGF-alpha-GIII-KDEL 2gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtctgacctg cccggtcgcc gccggtgaat 660gcgcgggccc ggcggacagc ggcgacgccc tgctggagcg caactatccc actggcgcgg 720agttcctcgg cgacggcggc gacgtcagct tcagcacccg cggcacgcag aactggacgg 780tggagcggct gctccaggcg caccgccaac tggaggagcg cggctatgtg ttcgtcggct 840accacggcac cttcctcgaa gcggcgcaaa gcatcgtctt cggcggggtg cgcgcgcgca 900gccaggacct cgacgcgatc tggcgcggtt tctatatcgc cggcgatccg gcgctggcct 960acggctacgc ccaggaccag gaacccgacg cacgcggccg gatccgcaac ggtgccctgc 1020tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta ccgcaccagc ctgaccctgg 1080ccgcgccgga ggcggcgggc gaggtcgaac ggctgatcgg ccatccgctg ccgctgcgcc 1140tggacgccat caccggcccc gaggaggaag gcgggcgcct ggagaccatt ctcggctggc 1200cgctggccga gcgcaccgtg gtgattccct cggcgatccc caccgacccg cgcaacgtcg 1260gcggcgacct cgacccgtcc agcatccccg acaaggaaca ggcgatcagc gccctgccgg 1320actacgccag ccagcccggc aaaccgccga aagatgaact gtaactcgag tagatctgta 1380ctctagaaag gagtcgttat gaaaaaaata acagggatta ttttattgct tcttgcagtc 1440attattctgt ctgcatgtca ggcaaactat atccgggatg ttcagggcgg gaccgtatct 1500ccgtcatcaa cagctgaagt gaccggatta gcaacgcagt aactgcaggc atgcaagctt 1560ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga 1620agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc 1680atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg 1740agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 1800tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc 1860ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 1920tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 1980aactcttttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 2040ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 2100gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 2160ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 2220gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2280agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 2340caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 2400gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 2460agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 2520gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 2580aatgaagcca taccaaacga cgagcgtgac accacgatgc ctacagcaat ggcaacaacg 2640ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 2700tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 2760tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 2820gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 2880atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 2940ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 3000aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 3060ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 3120ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 3180tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 3240cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 3300gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 3360gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 3420tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 3480ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 3540gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 3600ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 3660tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 3720ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 3780gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 3840acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 3900ctccttacgc atctgtgcgg tatttcacac cgcatatg 393833938DNAArtificial Sequencearabinose inducible plasmid, with start codon at 351, stop codon at 1364, and the colicin E3 lysis protein from 1399 to 1542 3gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtctgacctg cccggtcgcc gccggtgaat 660gcgcgggccc ggcggacagc ggcgacgccc tgctggagcg caactatccc actggcgcgg 720agttcctcgg cgacggcggc gacgtcagct tcagcacccg cggcacgcag aactggacgg 780tggagcggct gctccaggcg caccgccaac tggaggagcg cggctatgtg ttcgtcggct 840accacggcac cttcctcgaa gcggcgcaaa gcatcgtctt cggcggggtg cgcgcgcgca 900gccaggacct cgacgcgatc tggcgcggtt tctatatcgc cggcgatccg gcgctggcct 960acggctacgc ccaggaccag gaacccgacg cacgcggccg gatccgcaac ggtgccctgc 1020tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta ccgcaccagc ctgaccctgg 1080ccgcgccgga ggcggcgggc gaggtcgaac ggctgatcgg ccatccgctg ccgctgcgcc 1140tggacgccat caccggcccc gaggaggaag gcgggcgcct ggagaccatt ctcggctggc 1200cgctggccga gcgcaccgtg gtgattccct cggcgatccc caccgacccg cgcaacgtcg 1260gcggcgacct cgacccgtcc agcatccccg acaaggaaca ggcgatcagc gccctgccgg 1320actacgccag ccagcccggc aaaccgccga aagatgaact gtaactcgag tagatctgta 1380ctctagaaag gagtcgttat gaaaaaaata acagggatta ttttattgct tcttgcagtc 1440attattctgt ctgcatgtca ggcaaactat atccgggatg ttcagggcgg gaccgtatct 1500ccgtcatcaa cagctgaagt gaccggatta gcaacgcagt aactgcaggc atgcaagctt 1560ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga 1620agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc 1680atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg 1740agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 1800tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc 1860ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 1920tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 1980aactcttttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 2040ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 2100gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 2160ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 2220gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2280agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 2340caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 2400gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 2460agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 2520gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 2580aatgaagcca taccaaacga cgagcgtgac accacgatgc ctacagcaat ggcaacaacg 2640ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 2700tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 2760tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 2820gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 2880atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 2940ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 3000aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 3060ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 3120ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 3180tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 3240cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 3300gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 3360gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 3420tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 3480ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 3540gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 3600ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 3660tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 3720ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 3780gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 3840acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 3900ctccttacgc atctgtgcgg tatttcacac cgcatatg 393843711DNAArtificial SequenceModified TGF-alpha-GIII-KDEL 4gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtactggcgc ggagttcctc ggcgacggcg 660gcgacgtcag cttcagcacc cgcggcacgc agaactggac ggtggagcgg ctgctccagg 720cgcaccgcca actggaggag cgcggctatg tgttcgtcgg ctaccacggc accttcctcg 780aagcggcgca aagcatcgtc ttcggcgggg tgcgcgcgcg cagccaggac ctcgacgcga 840tctggcgcgg tttctatatc gccggcgatc cggcgctggc ctacggctac gcccaggacc 900aggaacccga cgcacgcggc cggatccgca acggtgccct gctgcgggtc tatgtgccgc 960gctcgagcct gccgggcttc taccgcacca gcctgaccct ggccgcgccg gaggcggcgg 1020gcgaggtcga acggctgatc ggccatccgc tgccgctgcg cctggacgcc atcaccggcc 1080ccgaggagga aggcgggcgc ctggagacca ttctcggctg gccgctggcc gagcgcaccg 1140tggtgattcc ctcggcgatc cccaccgacc cgcgcaacgt cggcggcgac ctcgacccgt 1200ccagcatccc cgacaaggaa caggcgatca gcgccctgcc ggactacgcc agccagcccg 1260gcaaaccgcc gaaagatgaa ctgtaactcg agtagatctg tactctagag tcgacctgca 1320ggcatgcaag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa 1380atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 1440cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 1500gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 1560aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 1620atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 1680gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 1740ttgcgtttct acaaactctt tttgtttatt tttctaaata cattcaaata tgtatccgct 1800catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 1860tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 1920tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1980ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 2040ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 2100cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 2160ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 2220tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 2280gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 2340ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctacagc 2400aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 2460acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 2520tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 2580cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 2640gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 2700taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 2760tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 2820cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2880ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2940accagcggtg gtttgtttgc cggatcaaga gctaccaact

ctttttccga aggtaactgg 3000cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 3060cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3120tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3180taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3240gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 3300agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 3360ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 3420acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 3480caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 3540tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 3600tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 3660gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat g 371154089DNAArtificial Sequencearabinose inducible plasmid expressing the construct with start codon at 351 and stop codon at 1664 5gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtggcagcct ggccgcgctg accgcgcacc 660aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc ggctgggaac 720aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg gcggcgcggc 780tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc ggcagcggcg 840gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc ctgaccctgg 900ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag gccggcgcgg 960ccaacgcccc ggcggacagc ggcgacgccc tgctggagcg caactatccc actggcgcgg 1020agttcctcgg cgacggcggc gacgtcagct tcagcacccg cggcacgcag aactggacgg 1080tggagcggct gctccaggcg caccgccaac tggaggagcg cggctatgtg ttcgtcggct 1140accacggcac cttcctcgaa gcggcgcaaa gcatcgtctt cggcggggtg cgcgcgcgca 1200gccaggacct cgacgcgatc tggcgcggtt tctatatcgc cggcgatccg gcgctggcct 1260acggctacgc ccaggaccag gaacccgacg cacgcggccg gatccgcaac ggtgccctgc 1320tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta ccgcaccagc ctgaccctgg 1380ccgcgccgga ggcggcgggc gaggtcgaac ggctgatcgg ccatccgctg ccgctgcgcc 1440tggacgccat caccggcccc gaggaggaag gcgggcgcct ggagaccatt ctcggctggc 1500cgctggccga gcgcaccgtg gtgattccct cggcgatccc caccgacccg cgcaacgtcg 1560gcggcgacct cgacccgtcc agcatccccg acaaggaaca ggcgatcagc gccctgccgg 1620actacgccag ccagcccggc aaaccgccga aagatgaact gtaactcgag tagatctgta 1680ctctagagtc gacctgcagg catgcaagct tggctgtttt ggcggatgag agaagatttt 1740cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg 1800cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag 1860cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa 1920aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 1980ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg 2040gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca 2100tcctgacgga tggccttttt gcgtttctac aaactctttt tgtttatttt tctaaataca 2160ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 2220aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 2280ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 2340gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 2400ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 2460ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 2520gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 2580aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 2640gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 2700aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 2760caccacgatg cctacagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 2820tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 2880acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2940gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 3000agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 3060gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 3120ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 3180taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3240agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3300aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3360ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 3420gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3480aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3540aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3600gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3660aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3720aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3780cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3840cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3900tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3960tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 4020ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 4080ccgcatatg 408964238DNAArtificial Sequencearabinose inducible plasmid expressing the construct with start codon at 351, a stop codon at 1664, and colicin E3 lysis protein from 1699 to 1842 6gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggctaaaa 360agacagctat cgcgattgca gtggcactgg ctggtttcgc taccgtagcg caggcagctg 420tggtgagcca ttttaacgat tgcccggata gccataccca gttttgcttt catggcacct 480gccgctttct ggtgcaggaa gataaaccgg cgtgcgtgtg ccatagcggt tatgtgggcg 540ctcgctgcga acatgcggat ctgctggccc atcaactagt gggcggtggt ggcagtggtg 600gcggcggctc tggtggtggc gggtcccggg gtggcagcct ggccgcgctg accgcgcacc 660aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc ggctgggaac 720aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg gcggcgcggc 780tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc ggcagcggcg 840gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc ctgaccctgg 900ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag gccggcgcgg 960ccaacgcccc ggcggacagc ggcgacgccc tgctggagcg caactatccc actggcgcgg 1020agttcctcgg cgacggcggc gacgtcagct tcagcacccg cggcacgcag aactggacgg 1080tggagcggct gctccaggcg caccgccaac tggaggagcg cggctatgtg ttcgtcggct 1140accacggcac cttcctcgaa gcggcgcaaa gcatcgtctt cggcggggtg cgcgcgcgca 1200gccaggacct cgacgcgatc tggcgcggtt tctatatcgc cggcgatccg gcgctggcct 1260acggctacgc ccaggaccag gaacccgacg cacgcggccg gatccgcaac ggtgccctgc 1320tgcgggtcta tgtgccgcgc tcgagcctgc cgggcttcta ccgcaccagc ctgaccctgg 1380ccgcgccgga ggcggcgggc gaggtcgaac ggctgatcgg ccatccgctg ccgctgcgcc 1440tggacgccat caccggcccc gaggaggaag gcgggcgcct ggagaccatt ctcggctggc 1500cgctggccga gcgcaccgtg gtgattccct cggcgatccc caccgacccg cgcaacgtcg 1560gcggcgacct cgacccgtcc agcatccccg acaaggaaca ggcgatcagc gccctgccgg 1620actacgccag ccagcccggc aaaccgccga aagatgaact gtaactcgag tagatctgta 1680ctctagaaag gagtcgttat gaaaaaaata acagggatta ttttattgct tcttgcagtc 1740attattctgt ctgcatgtca ggcaaactat atccgggatg ttcagggcgg gaccgtatct 1800ccgtcatcaa cagctgaagt gaccggatta gcaacgcagt aactgcaggc atgcaagctt 1860ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga 1920agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc 1980atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg 2040agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 2100tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc 2160ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 2220tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 2280aactcttttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 2340ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 2400gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 2460ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 2520gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2580agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 2640caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 2700gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 2760agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 2820gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 2880aatgaagcca taccaaacga cgagcgtgac accacgatgc ctacagcaat ggcaacaacg 2940ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 3000tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 3060tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 3120gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 3180atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 3240ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 3300aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 3360ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 3420ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 3480tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 3540cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 3600gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 3660gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 3720tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 3780ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 3840gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 3900ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 3960tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 4020ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 4080gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 4140acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 4200ctccttacgc atctgtgcgg tatttcacac cgcatatg 423874770DNAArtificial Sequencearabinose inducible plasmid expressing the construct with cldtB start codon at 351, stop codon at 1163, pltB start at 1197, stop at 1610, and pltA 1627-2355 7gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggtcaaaa 360aacctgtttt tttccttctg accatgatca tctgcagcta tatttctttt gcctgcgcta 420atatcagtga ctacaaagtt atgacctgga atcttcaggg ctcttcagca tctacagaaa 480gtaaatggaa tgtcaatgtc agacagcttt taagcggtac tgccggtgtg gatattctta 540tggtacagga ggccggtgct gttcccacct cagcggttcc taccggacga catattcagc 600cttttggagt gggtattccc attgatgaat acacctggaa tctcggaacc accagccgtc 660aggatataag atatatctac tactcggcta ttgatgttgg agcacgccgt gttaatctgg 720caatagtttc cagacaaaga gcggataatg tttatgtctt gcgtccgaca actgtcgcat 780ctcgccccgt cattggcatc ggactgggta atgatgtttt tctgacagcg cacgcactgg 840ctagtggagg tccggatgct gcagctattg tcagggttac cattaatttt tttagacaac 900ctcagatgcg gcatttatcc tggtttcttg ccggggattt taatcgcagc ccagacagac 960ttgaaaatga cctgatgact gagcatctgg aacgagttgt agccgtactc gcacctacag 1020aacccacgca aattagcggt ggtattttag attatggggt cattgtcgat cgagcacctt 1080attcacaaag ggtcgaagca ttacgtaatc cacaactcgc ttctgatcat tatcccgtag 1140cctttttggc acgaagctgt taatgatcta gataagtcga cgtacaggag agtagtatgt 1200atataaataa gtttgtgcct gtttatacat tattaattct catttattct tttaatgcca 1260gcgctgagtg gacaggagat aatacgaacg cctattactc agacgaagtt atcagtgaat 1320tacatgttgg tcagatagat actagtcctt atttttgcat aaaaacggtt aaagctaacg 1380gtagtggtac accagttgtt gcatgtgcgg tatcaaagca gagcatatgg gcgccctcct 1440ttaaagaact tcttgatcag gcaagatatt tttacagtac agggcaatcc gtaaggattc 1500atgttcaaaa aaatatctgg acctatccgc tttttgttaa taccttttca gcaaatgctc 1560ttgtgggact atcatcgtgc agtgcgacac aatgctttgg acccaagtaa gaggggagaa 1620gaaataatga aaaagttaat attcttaacc ttatctatag ttagctttaa taactatgct 1680gtagattttg tgtatcgtgt ggactcaacc ccgccggacg ttatttttcg cgatgggttt 1740tcactacttg ggtataaccg taactttcag caatttatta gtggaaggtc atgtagtggt 1800ggaagtagtg acagtcgcta tattgcaaca acctcaagtg ttaatcaaac atatgctata 1860gccagagcgt actattctcg ctcaacattc aaaggtaatt tatacagata tcagattcgt 1920gcagataata atttctacag cttgctccca tccatcacct atctggaaac gcaaggtggt 1980cactttaatg cttatgaaaa aacgatgatg cgattgcaaa gagagtatgt ttccacatta 2040tctattttac ccgagaatat tcaaaaggcc gtggcgctag tttatgatag cgcaaccggt 2100ctggtaaagg atggtgtaag cacaatgaat gccagttatt taggtttaag cactacgtct 2160aatcctggtg tgataccttt tcttccggaa ccgcagacgt atacccaaca acgaattgat 2220gcattcggcc cattaataag ttcatgcttt tcaataggta gcgtatgtca gtcacatcga 2280gggcaaagag ctgacgtata caacatgtct ttttatgatg caagacctgt aatagaactt 2340atactttcta aataaatgaa acttacctat gttgccgcat gcaagctagc ttggctgttt 2400tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct 2460gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa 2520ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg 2580gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 2640tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga 2700acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 2760atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt 2820ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 2880aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 2940tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 3000agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 3060cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3120taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg 3180tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 3240tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 3300cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 3360gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3420cataccaaac gacgagcgtg acaccacgat gcctacagca atggcaacaa cgttgcgcaa 3480actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 3540ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 3600tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 3660tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 3720acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 3780ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 3840ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 3900ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 3960gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 4020ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 4080aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 4140gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 4200gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 4260aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 4320cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 4380tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 4440ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 4500atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 4560cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 4620ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 4680gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac 4740gcatctgtgc ggtatttcac accgcatatg 4770815DNAArtificial SequenceLinker x3 8ggggsggggs ggggs 1594851DNAArtificial Sequencearabinose inducible plasmid with a cldtB start codon at 351 through codon at 1160 (without a stop), FLAG from 1170-1193, and GGGGS(x3) from 1194-1238, pltB start at 1278 and stop at 1691 and pltA 1708-2436 9gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggtcaaaa 360aacctgtttt tttccttctg

accatgatca tctgcagcta tatttctttt gcctgcgcta 420atatcagtga ctacaaagtt atgacctgga atcttcaggg ctcttcagca tctacagaaa 480gtaaatggaa tgtcaatgtc agacagcttt taagcggtac tgccggtgtg gatattctta 540tggtacagga ggccggtgct gttcccacct cagcggttcc taccggacga catattcagc 600cttttggagt gggtattccc attgatgaat acacctggaa tctcggaacc accagccgtc 660aggatataag atatatctac tactcggcta ttgatgttgg agcacgccgt gttaatctgg 720caatagtttc cagacaaaga gcggataatg tttatgtctt gcgtccgaca actgtcgcat 780ctcgccccgt cattggcatc ggactgggta atgatgtttt tctgacagcg cacgcactgg 840ctagtggagg tccggatgct gcagctattg tcagggttac cattaatttt tttagacaac 900ctcagatgcg gcatttatcc tggtttcttg ccggggattt taatcgcagc ccagacagac 960ttgaaaatga cctgatgact gagcatctgg aacgagttgt agccgtactc gcacctacag 1020aacccacgca aattagcggt ggtattttag attatggggt cattgtcgat cgagcacctt 1080attcacaaag ggtcgaagca ttacgtaatc cacaactcgc ttctgatcat tatcccgtag 1140cctttttggc acgaagctgt ataccaggtg attataaaga tgacgatgac aaaggcggtg 1200gcggtagcgg cggtggcggt tctggcggtg gcggtagtaa gcttgggtct agataggtcg 1260acgtacagga gagtagtatg tatataaata agtttgtgcc tgtttataca ttattaattc 1320tcatttattc ttttaatgcc agcgctgagt ggacaggaga taatacgaac gcctattact 1380cagacgaagt tatcagtgaa ttacatgttg gtcagataga tactagtcct tatttttgca 1440taaaaacggt taaagctaac ggtagtggta caccagttgt tgcatgtgcg gtatcaaagc 1500agagcatatg ggcgccctcc tttaaagaac ttcttgatca ggcaagatat ttttacagta 1560cagggcaatc cgtaaggatt catgttcaaa aaaatatctg gacctatccg ctttttgtta 1620ataccttttc agcaaatgct cttgtgggac tatcatcgtg cagtgcgaca caatgctttg 1680gacccaagta agaggggaga agaaataatg aaaaagttaa tattcttaac cttatctata 1740gttagcttta ataactatgc tgtagatttt gtgtatcgtg tggactcaac cccgccggac 1800gttatttttc gcgatgggtt ttcactactt gggtataacc gtaactttca gcaatttatt 1860agtggaaggt catgtagtgg tggaagtagt gacagtcgct atattgcaac aacctcaagt 1920gttaatcaaa catatgctat agccagagcg tactattctc gctcaacatt caaaggtaat 1980ttatacagat atcagattcg tgcagataat aatttctaca gcttgctccc atccatcacc 2040tatctggaaa cgcaaggtgg tcactttaat gcttatgaaa aaacgatgat gcgattgcaa 2100agagagtatg tttccacatt atctatttta cccgagaata ttcaaaaggc cgtggcgcta 2160gtttatgata gcgcaaccgg tctggtaaag gatggtgtaa gcacaatgaa tgccagttat 2220ttaggtttaa gcactacgtc taatcctggt gtgatacctt ttcttccgga accgcagacg 2280tatacccaac aacgaattga tgcattcggc ccattaataa gttcatgctt ttcaataggt 2340agcgtatgtc agtcacatcg agggcaaaga gctgacgtat acaacatgtc tttttatgat 2400gcaagacctg taatagaact tatactttct aaataaatga aacttaccta tgttgccgca 2460tgcaagctag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa 2520atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 2580cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 2640gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 2700aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 2760atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 2820gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 2880ttgcgtttct acaaactctt tttgtttatt tttctaaata cattcaaata tgtatccgct 2940catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 3000tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 3060tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 3120ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 3180ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 3240cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 3300ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 3360tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 3420gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 3480ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctacagc 3540aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 3600acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 3660tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 3720cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 3780gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 3840taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3900tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3960cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 4020ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 4080accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 4140cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 4200cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4260tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4320taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4380gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4440agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4500ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4560acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4620caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4680tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4740tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 4800gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat g 4851105250DNAArtificial Sequencearabinose inducible plasmid expressing the TAT-apoptin construct as a cldtB fusion, with TAT-apoptin inserted in-frame within the HindIII and Xbal sites introduced into typhoid toxin cldtB, with the TAT-apoptin coding sequence from 1245-1646 10gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggtcaaaa 360aacctgtttt tttccttctg accatgatca tctgcagcta tatttctttt gcctgcgcta 420atatcagtga ctacaaagtt atgacctgga atcttcaggg ctcttcagca tctacagaaa 480gtaaatggaa tgtcaatgtc agacagcttt taagcggtac tgccggtgtg gatattctta 540tggtacagga ggccggtgct gttcccacct cagcggttcc taccggacga catattcagc 600cttttggagt gggtattccc attgatgaat acacctggaa tctcggaacc accagccgtc 660aggatataag atatatctac tactcggcta ttgatgttgg agcacgccgt gttaatctgg 720caatagtttc cagacaaaga gcggataatg tttatgtctt gcgtccgaca actgtcgcat 780ctcgccccgt cattggcatc ggactgggta atgatgtttt tctgacagcg cacgcactgg 840ctagtggagg tccggatgct gcagctattg tcagggttac cattaatttt tttagacaac 900ctcagatgcg gcatttatcc tggtttcttg ccggggattt taatcgcagc ccagacagac 960ttgaaaatga cctgatgact gagcatctgg aacgagttgt agccgtactc gcacctacag 1020aacccacgca aattagcggt ggtattttag attatggggt cattgtcgat cgagcacctt 1080attcacaaag ggtcgaagca ttacgtaatc cacaactcgc ttctgatcat tatcccgtag 1140cctttttggc acgaagctgt ataccaggtg attataaaga tgacgatgac aaaggcggtg 1200gcggtagcgg cggtggcggt tctggcggtg gcggtagtaa gcttatggca tacggtcgca 1260agaaacgtcg tcaacgccgc cgcatgaacg ccctgcaaga agacacgccg ccgggtccga 1320gcacggtttt tcgtccgccg accagctctc gcccgctgga aacgccgcat tgccgtgaaa 1380ttcgcatcgg cattgcaggt atcaccatta cgctgtctct gtgcggctgt gcaaacgcac 1440gtgcaccgac cctgcgctcc gctacggcgg ataacagtga atccaccggt tttaaaaatg 1500tgccggatct gcgtacggac cagccgaagc cgccgtcaaa aaagcgttcg tgtgacccga 1560gcgaatatcg cgttagcgaa ctgaaagaat ctctgattac cacgaccccg tcccgtccgc 1620gcaccgcaaa acgccgcatc cgtctgtcta gataggtcga cgtacaggag agtagtatgt 1680atataaataa gtttgtgcct gtttatacat tattaattct catttattct tttaatgcca 1740gcgctgagtg gacaggagat aatacgaacg cctattactc agacgaagtt atcagtgaat 1800tacatgttgg tcagatagat actagtcctt atttttgcat aaaaacggtt aaagctaacg 1860gtagtggtac accagttgtt gcatgtgcgg tatcaaagca gagcatatgg gcgccctcct 1920ttaaagaact tcttgatcag gcaagatatt tttacagtac agggcaatcc gtaaggattc 1980atgttcaaaa aaatatctgg acctatccgc tttttgttaa taccttttca gcaaatgctc 2040ttgtgggact atcatcgtgc agtgcgacac aatgctttgg acccaagtaa gaggggagaa 2100gaaataatga aaaagttaat attcttaacc ttatctatag ttagctttaa taactatgct 2160gtagattttg tgtatcgtgt ggactcaacc ccgccggacg ttatttttcg cgatgggttt 2220tcactacttg ggtataaccg taactttcag caatttatta gtggaaggtc atgtagtggt 2280ggaagtagtg acagtcgcta tattgcaaca acctcaagtg ttaatcaaac atatgctata 2340gccagagcgt actattctcg ctcaacattc aaaggtaatt tatacagata tcagattcgt 2400gcagataata atttctacag cttgctccca tccatcacct atctggaaac gcaaggtggt 2460cactttaatg cttatgaaaa aacgatgatg cgattgcaaa gagagtatgt ttccacatta 2520tctattttac ccgagaatat tcaaaaggcc gtggcgctag tttatgatag cgcaaccggt 2580ctggtaaagg atggtgtaag cacaatgaat gccagttatt taggtttaag cactacgtct 2640aatcctggtg tgataccttt tcttccggaa ccgcagacgt atacccaaca acgaattgat 2700gcattcggcc cattaataag ttcatgcttt tcaataggta gcgtatgtca gtcacatcga 2760gggcaaagag ctgacgtata caacatgtct ttttatgatg caagacctgt aatagaactt 2820atactttcta aataaatgaa acttacctat gttgccgcat gcaagctagc ttggctgttt 2880tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct 2940gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa 3000ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg 3060gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 3120tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga 3180acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 3240atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt 3300ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 3360aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 3420tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 3480agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 3540cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3600taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg 3660tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 3720tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 3780cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 3840gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3900cataccaaac gacgagcgtg acaccacgat gcctacagca atggcaacaa cgttgcgcaa 3960actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 4020ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 4080tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 4140tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 4200acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 4260ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 4320ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 4380ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 4440gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 4500ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 4560aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 4620gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 4680gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 4740aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 4800cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 4860tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 4920ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 4980atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 5040cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 5100ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 5160gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac 5220gcatctgtgc ggtatttcac accgcatatg 52501139DNAArtificial Sequenceparatyphi A NcoI_F1 11tacgccatgg tcaaaaaacc tgtttttttc cttctgacc 391243DNAArtificial SequencePCR primer HindIII_Xbal Clal-R1 12cgatccctct agacccaagc ttgtcactga tattagcgca ggc 431349DNAArtificial SequencePCR primer HindIII_Xbal_Clal-F1 13gcttgggtct agagggatcg attacaaagt tatgacctgg aatcttcag 491439DNAArtificial SequencePCR primer cldtB_stop_Xbal_Sall-R1 14gatcgtcgac ttatctagat cattaacagc ttcgtgcca 39154794DNAArtificial Sequence13-350 = arabinose promoter; 351-1187 = cldtB; 1210-1213 =RBS pltB; 1221-1634 = pltB; 1651-2379 = pltA 15gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggtcaaaa 360aacctgtttt tttccttctg accatgatca tctgcagcta tatttctttt gcctgcgcta 420atatcagtga caagcttggg tctagaggga tcgattacaa agttatgacc tggaatcttc 480agggctcttc agcatctaca gaaagtaaat ggaatgtcaa tgtcagacag cttttaagcg 540gtactgccgg tgtggatatt cttatggtac aggaggccgg tgctgttccc acctcagcgg 600ttcctaccgg acgacatatt cagccttttg gagtgggtat tcccattgat gaatacacct 660ggaatctcgg aaccaccagc cgtcaggata taagatatat ctactactcg gctattgatg 720ttggagcacg ccgtgttaat ctggcaatag tttccagaca aagagcggat aatgtttatg 780tcttgcgtcc gacaactgtc gcatctcgcc ccgtcattgg catcggactg ggtaatgatg 840tttttctgac agcgcacgca ctggctagtg gaggtccgga tgctgcagct attgtcaggg 900ttaccattaa tttttttaga caacctcaga tgcggcattt atcctggttt cttgccgggg 960attttaatcg cagcccagac agacttgaaa atgacctgat gactgagcat ctggaacgag 1020ttgtagccgt actcgcacct acagaaccca cgcaaattag cggtggtatt ttagattatg 1080gggtcattgt cgatcgagca ccttattcac aaagggtcga agcattacgt aatccacaac 1140tcgcttctga tcattatccc gtagcctttt tggcacgaag ctgttaatga tctagataag 1200tcgacgtaca ggagagtagt atgtatataa ataagtttgt gcctgtttat acattattaa 1260ttctcattta ttcttttaat gccagcgctg agtggacagg agataatacg aacgcctatt 1320actcagacga agttatcagt gaattacatg ttggtcagat agatactagt ccttattttt 1380gcataaaaac ggttaaagct aacggtagtg gtacaccagt tgttgcatgt gcggtatcaa 1440agcagagcat atgggcgccc tcctttaaag aacttcttga tcaggcaaga tatttttaca 1500gtacagggca atccgtaagg attcatgttc aaaaaaatat ctggacctat ccgctttttg 1560ttaatacctt ttcagcaaat gctcttgtgg gactatcatc gtgcagtgcg acacaatgct 1620ttggacccaa gtaagagggg agaagaaata atgaaaaagt taatattctt aaccttatct 1680atagttagct ttaataacta tgctgtagat tttgtgtatc gtgtggactc aaccccgccg 1740gacgttattt ttcgcgatgg gttttcacta cttgggtata accgtaactt tcagcaattt 1800attagtggaa ggtcatgtag tggtggaagt agtgacagtc gctatattgc aacaacctca 1860agtgttaatc aaacatatgc tatagccaga gcgtactatt ctcgctcaac attcaaaggt 1920aatttataca gatatcagat tcgtgcagat aataatttct acagcttgct cccatccatc 1980acctatctgg aaacgcaagg tggtcacttt aatgcttatg aaaaaacgat gatgcgattg 2040caaagagagt atgtttccac attatctatt ttacccgaga atattcaaaa ggccgtggcg 2100ctagtttatg atagcgcaac cggtctggta aaggatggtg taagcacaat gaatgccagt 2160tatttaggtt taagcactac gtctaatcct ggtgtgatac cttttcttcc ggaaccgcag 2220acgtataccc aacaacgaat tgatgcattc ggcccattaa taagttcatg cttttcaata 2280ggtagcgtat gtcagtcaca tcgagggcaa agagctgacg tatacaacat gtctttttat 2340gatgcaagac ctgtaataga acttatactt tctaaataaa tgaaacttac ctatgttgcc 2400gcatgcaagc tagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat 2460taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt 2520ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt 2580ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt 2640cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga 2700caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag 2760gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc 2820tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa atatgtatcc 2880gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 2940tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3000tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 3060gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 3120acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 3180tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 3240gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 3300tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 3360accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 3420ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctac 3480agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 3540gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 3600ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 3660tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 3720ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 3780gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 3840acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 3900aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 3960atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4020gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4080tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 4140ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 4200ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 4260ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 4320aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 4380cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 4440gagggagctt ccagggggaa

acgcctggta tctttatagt cctgtcgggt ttcgccacct 4500ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 4560cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 4620tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 4680cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 4740cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatg 4794164902DNAArtificial Sequence13-350 = arabinose promoter; 351-1295 = cldtB; 1318-1321 =RBS pltB; 1329-1742 = pltB; 1759-2487 = pltA 16gggggcggcc gcaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct 60tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac 120aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa 180aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca 240taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 300cgtttttttg ggctagcgaa ttcgagctcg gtacccagga ggaattcacc atggtcaaaa 360aacctgtttt tttccttctg accatgatca tctgcagcta tatttctttt gcctgcgcta 420atatcagtga caagcttggg tctagagact acaaagacca tgacggtgat tataaagatc 480atgatatcga ctacaaagat gacgacgata aaggcggtgg cggatccggc ggtggcggtt 540ctggcggtgg cggtagtatc gattacaaag ttatgacctg gaatcttcag ggctcttcag 600catctacaga aagtaaatgg aatgtcaatg tcagacagct tttaagcggt actgccggtg 660tggatattct tatggtacag gaggccggtg ctgttcccac ctcagcggtt cctaccggac 720gacatattca gccttttgga gtgggtattc ccattgatga atacacctgg aatctcggaa 780ccaccagccg tcaggatata agatatatct actactcggc tattgatgtt ggagcacgcc 840gtgttaatct ggcaatagtt tccagacaaa gagcggataa tgtttatgtc ttgcgtccga 900caactgtcgc atctcgcccc gtcattggca tcggactggg taatgatgtt tttctgacag 960cgcacgcact ggctagtgga ggtccggatg ctgcagctat tgtcagggtt accattaatt 1020tttttagaca acctcagatg cggcatttat cctggtttct tgccggggat tttaatcgca 1080gcccagacag acttgaaaat gacctgatga ctgagcatct ggaacgagtt gtagccgtac 1140tcgcacctac agaacccacg caaattagcg gtggtatttt agattatggg gtcattgtcg 1200atcgagcacc ttattcacaa agggtcgaag cattacgtaa tccacaactc gcttctgatc 1260attatcccgt agcctttttg gcacgaagct gttaatgatc tagataagtc gacgtacagg 1320agagtagtat gtatataaat aagtttgtgc ctgtttatac attattaatt ctcatttatt 1380cttttaatgc cagcgctgag tggacaggag ataatacgaa cgcctattac tcagacgaag 1440ttatcagtga attacatgtt ggtcagatag atactagtcc ttatttttgc ataaaaacgg 1500ttaaagctaa cggtagtggt acaccagttg ttgcatgtgc ggtatcaaag cagagcatat 1560gggcgccctc ctttaaagaa cttcttgatc aggcaagata tttttacagt acagggcaat 1620ccgtaaggat tcatgttcaa aaaaatatct ggacctatcc gctttttgtt aatacctttt 1680cagcaaatgc tcttgtggga ctatcatcgt gcagtgcgac acaatgcttt ggacccaagt 1740aagaggggag aagaaataat gaaaaagtta atattcttaa ccttatctat agttagcttt 1800aataactatg ctgtagattt tgtgtatcgt gtggactcaa ccccgccgga cgttattttt 1860cgcgatgggt tttcactact tgggtataac cgtaactttc agcaatttat tagtggaagg 1920tcatgtagtg gtggaagtag tgacagtcgc tatattgcaa caacctcaag tgttaatcaa 1980acatatgcta tagccagagc gtactattct cgctcaacat tcaaaggtaa tttatacaga 2040tatcagattc gtgcagataa taatttctac agcttgctcc catccatcac ctatctggaa 2100acgcaaggtg gtcactttaa tgcttatgaa aaaacgatga tgcgattgca aagagagtat 2160gtttccacat tatctatttt acccgagaat attcaaaagg ccgtggcgct agtttatgat 2220agcgcaaccg gtctggtaaa ggatggtgta agcacaatga atgccagtta tttaggttta 2280agcactacgt ctaatcctgg tgtgatacct tttcttccgg aaccgcagac gtatacccaa 2340caacgaattg atgcattcgg cccattaata agttcatgct tttcaatagg tagcgtatgt 2400cagtcacatc gagggcaaag agctgacgta tacaacatgt ctttttatga tgcaagacct 2460gtaatagaac ttatactttc taaataaatg aaacttacct atgttgccgc atgcaagcta 2520gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg 2580cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga 2640ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca 2700tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 2760cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg 2820gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat 2880aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc 2940tacaaactct ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 3000ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 3060ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 3120aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 3180actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 3240gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 3300agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 3360cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 3420catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 3480aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 3540gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctacag caatggcaac 3600aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 3660agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 3720ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 3780actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 3840aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 3900gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 3960atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 4020tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 4080tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 4140ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 4200agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 4260ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 4320tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 4380gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 4440cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 4500ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 4560agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 4620tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 4680ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 4740ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 4800ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta 4860ttttctcctt acgcatctgt gcggtatttc acaccgcata tg 4902173560DNAArtificial Sequencetetracyclin-inducible YebFSFTI, start codon begins at 724 and the stop codon ends at 1158 17atatgggggc ggccgcctaa gacccacttt cacatttaag ttgtttttct aatccgcata 60taatcaattc aaggccgaat aagaaggctg gctctgcacc ttggtgttca aataattcga 120tagcttgtcg taataatgcc ggcatactat cagtagtagg tgtttccctt tcttctttag 180cgacttgatg ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga 240gtgcatataa tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt 300cgagagtttc atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc 360gatgacttag taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc 420tttccccttc taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg 480cgtcgagcaa agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccca 540gtttctgggc gagtttacgg gtttttaaac cttcgattcc gacctcatta agcagctcta 600atgcgctgtt aatcacttta cttttatcta atctggacat cataaattcc taatttttgt 660tgacactcta tcattgatag agttatttta ccactcccta tcagtgatag agaaaagtga 720accatggcta aaaaaagagg ggcgttttta gggctgttgt tggtttctgc ctgcgcatca 780gttttcgctg ccaataatga aaccagcaag tcggtcactt tcccaaagtg tgaagatctg 840gatgctgccg gaattgccgc gagcgtaaaa cgtgattatc aacaaaatcg cgtggcgcgt 900tgggcagatg atcaaaaaat tgtcggtcag gccgatcccg tggcttgggt cagtttgcag 960gacattcagg gtaaagatga taaatggtca gtaccgctaa ccgtgcgtgg taaaagtgcc 1020gatattcatt accaggtcag cgtggactgc aaagcgggaa tggcggaata tcagcggcgt 1080ctcgaggacg atgacgataa gggtaccctg aaaggccgct gcaccaaaag cattccgccg 1140atttgctttc cggattagtc tagagtcgac ctgcaggcat gcaagcttgg ctgttttggc 1200ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata 1260aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca 1320gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac 1380tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg 1440ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt 1500tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca 1560aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctctttttgt 1620ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 1680cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 1740cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 1800aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 1860ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 1920gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc 1980cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2040acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2100gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2160aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2220ccaaacgacg agcgtgacac cacgatgcct acagcaatgg caacaacgtt gcgcaaacta 2280ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2340gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2400aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2460aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2520aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 2580gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 2640gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 2700tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 2760gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 2820caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 2880actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 2940acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 3000cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 3060gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 3120cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 3180gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 3240tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 3300tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 3360gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 3420aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 3480agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 3540ctgtgcggta tttcacaccg 3560183249DNAArtificial Sequencetetracycline inducible OmpASFTI fusion, start codon begins at 725 and stop codon ends at 1008 18catatggggg cggccgccta agacccactt tcacatttaa gttgtttttc taatccgcat 60ataatcaatt caaggccgaa taagaaggct ggctctgcac cttggtgttc aaataattcg 120atagcttgtc gtaataatgc cggcatacta tcagtagtag gtgtttccct ttcttcttta 180gcgacttgat gctcttgatc ttccaatacg caacctaaag taaaatgccc cacagcgctg 240agtgcatata atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc taattgattt 300tcgagagttt catactgttt ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg 360cgatgactta gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa atcttgccag 420ctttcccctt ctaaagggca aaagtgagta tggtgcctat ctaacatctc aatggctaag 480gcgtcgagca aagcccgctt attttttaca tgccaataca atgtaggctg ctctacaccc 540agtttctggg cgagtttacg ggtttttaaa ccttcgattc cgacctcatt aagcagctct 600aatgcgctgt taatcacttt acttttatct aatctggaca tcataaattc ctaatttttg 660ttgacactct atcattgata gagttatttt accactccct atcagtgata gagaaaagtg 720aaccatggct aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt 780agcgcaggcc gctccgaaag atggccgctg caccaaaagc attccgccga tttgctttcc 840ggattagtct agagtcgacc tgcaggcatg caagcttggc tgttttggcg gatgagagaa 900gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt 960gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg 1020ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc 1080aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg 1140tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac 1200ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga 1260aggccatcct gacggatggc ctttttgcgt ttctacaaac tctttttgtt tatttttcta 1320aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1380ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1440ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1500agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1560tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1620tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1680ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1740gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1800acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1860tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1920gcgtgacacc acgatgccta cagcaatggc aacaacgttg cgcaaactat taactggcga 1980actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 2040aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2100cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2160tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2220cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2280tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2340ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2400ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2460cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2520aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2580agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2640tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2700ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2760cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2820atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2880ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2940tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3000gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3060gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3120cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3180gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3240ttcacaccg 3249193398DNAArtificial Sequencetetracycline inducible OmpASFTI fusion, start codon at 725, stop codon at 1008, followed by a Shine Delgarno sequence (RBS) and coding sequence for E3 Lysis sequence, start codon at 865, stop codon at 1008 19catatggggg cggccgccta agacccactt tcacatttaa gttgtttttc taatccgcat 60ataatcaatt caaggccgaa taagaaggct ggctctgcac cttggtgttc aaataattcg 120atagcttgtc gtaataatgc cggcatacta tcagtagtag gtgtttccct ttcttcttta 180gcgacttgat gctcttgatc ttccaatacg caacctaaag taaaatgccc cacagcgctg 240agtgcatata atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc taattgattt 300tcgagagttt catactgttt ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg 360cgatgactta gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa atcttgccag 420ctttcccctt ctaaagggca aaagtgagta tggtgcctat ctaacatctc aatggctaag 480gcgtcgagca aagcccgctt attttttaca tgccaataca atgtaggctg ctctacaccc 540agtttctggg cgagtttacg ggtttttaaa ccttcgattc cgacctcatt aagcagctct 600aatgcgctgt taatcacttt acttttatct aatctggaca tcataaattc ctaatttttg 660ttgacactct atcattgata gagttatttt accactccct atcagtgata gagaaaagtg 720aaccatggct aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt 780agcgcaggcc gctccgaaag atggccgctg caccaaaagc attccgccga tttgctttcc 840ggattagtct agaaaggagt cgttatgaaa aaaataacag ggattatttt attgcttctt 900gcagtcatta ttctgtctgc atgtcaggca aactatatcc gggatgttca gggcgggacc 960gtatctccgt catcaacagc tgaagtgacc ggattagcaa cgcagtaact gcaggcatgc 1020aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa 1080cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 1140gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 1200catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 1260ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 1320gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 1380ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 1440tctacaaact ctttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 1500caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 1560ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 1620gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 1680gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 1740atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg 1800caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 1860gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 1920accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 1980ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 2040gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctac agcaatggca 2100acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 2160atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 2220ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 2280gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac

ggggagtcag 2340gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 2400tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 2460taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 2520cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 2580gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 2640gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 2700agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 2760aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 2820agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 2880cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 2940accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 3000aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 3060ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 3120cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 3180gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 3240tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 3300agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 3360tattttctcc ttacgcatct gtgcggtatt tcacaccg 33982011PRTArtificial SequenceHIV TAT protein amino acids 47-57 20Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10219PRTArtificial Sequencelymphoid tissue targeting peptide 21Cys Ala Tyr His Arg Leu Arg Arg Cys1 52212PRTArtificial SequencePeptide from Clostridium 22Cys Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn1 5 10235PRTArtificial Sequencenative C-terminal signal of ToxA sequence 23Arg Glu Asp Leu Lys1 5