Chimeric protein toxins for expression by therapeutic bacteria

Abstract

Bacteria with tumor-targeting capability express, surface displayed, secreted and/or released modified chimeric therapeutic proteins with enhanced therapeutic activity against a neoplastic tissue including solid tumors, lymphomas and leukemias. The bacteria may be attenuated, non-pathogenic, low pathogenic or a probiotic. The chimeric proteins may be protease sensitive and may optionally be further accompanied by co-expression of a secreted protease inhibitor as a separate molecule or as a fusion.

Claims

1. A chimeric protease inhibitor comprising: a bacterial secretion signal peptide comprising YebF; and a serine protease inhibitor peptide comprising sunflower trypsin inhibitor; wherein the bacterial secretion signal peptide and the serine protease inhibitor peptide are fused to form a chimeric peptide.

2. The chimeric protease inhibitor according to claim 1, wherein the chimeric peptide has a sequence that encodes the nucleotide sequence of SEQ ID NO: 017.

3. The chimeric protease inhibitor according to claim 1, in a purified form.

4. The chimeric protease inhibitor according to claim 1, in combination with a live genetically engineered bacterium comprising a genetic sequence encoding the chimeric peptide and an inducible promoter adapted to promote transcription of the chimeric protease inhibitor within the genetically engineered bacterium.

5. The chimeric protease inhibitor according to claim 4, wherein the genetically engineered bacterium is alive within a pharmaceutically acceptable dosage form.

6. The chimeric protease inhibitor according to claim 1, in a pharmaceutically acceptable formulation further comprising a cytotoxic peptide comprising a chimeric cytolethal distending toxin:apoptin peptide degradable by a protease inhibited by the serine protease inhibitor.

7. The chimeric protease inhibitor according to claim 1, further comprising a chimeric Pseudomonas ToxA therapeutic molecule peptide degradable by a protease inhibited by the serine protease inhibitor.

8. The chimeric protease inhibitor according to claim 1, in a pharmaceutically effective dosage form comprising at least 10.sup.9 live genetically engineered bacteria that produce and secrete the chimeric protease inhibitor.

9. A chimeric protease inhibitor comprising a bacterial secretion signal comprising YebF, linked through at least one amino acid to a serine protease inhibitor peptide sequence comprising sunflower trypsin inhibitor.

10. The chimeric protease inhibitor according to claim 9, transcribed from a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 017.

11. The chimeric protease inhibitor according to claim 9, in combination with a live genetically engineered bacterium in a pharmaceutically acceptable formulation, the live genetically engineered bacterium comprising an inducible promoter for inducing production of the chimeric protease inhibitor by the live genetically engineered bacterium.

12. The chimeric protease inhibitor according to claim 9, in combination with a live genetically engineered bacterium in a pharmaceutically acceptable formulation, the live genetically engineered bacterium comprising a nucleic acid which encodes a reorganized polycistronic cytolethal distending toxin fusion expressed or secreted in an absence of sty and ttsa.

13. The chimeric protease inhibitor according to claim 9, in combination with a live genetically engineered bacterium in a pharmaceutically acceptable formulation, the live genetically engineered bacterium comprising a nucleic acid which encodes a modified chimeric cytolethal distending toxin:apoptin fusion expressed or secreted in an absence of in an absence of sty and ttsa.

14. The chimeric protease inhibitor according to claim 9, in combination with a live genetically engineered bacterium in a pharmaceutically acceptable formulation, the live genetically engineered bacterium comprising a nucleic acid which encodes a functionally active modified chimeric Pseudomonas ToxA therapeutic molecule.

15. The chimeric protease inhibitor according to claim 9, in combination with a live genetically engineered bacterium in a pharmaceutically acceptable formulation, the live genetically engineered bacterium being a tumor targeting bacterium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

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

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

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

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

(13) 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

(14) 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.

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

(16) Targeting Ligands

(17) 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, MA; 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- (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 (v6), 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, EGF, EGFR-binding peptides and other, non-limiting, peptides. In preferred embodiments, the peptides are affibody against HER2, H10 DARPIN against HER2, TGF, EGF, EGFR-binding peptides.

(18) 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.

(19) FIG. 1A shows a chimera containing an OmpA secretion signal, TGF targeting domain, a GGGGS (x3) (i.e., GGGGSGGGGSGGGGS, 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).

(20) FIG. 1B shows a chimera containing an OmpA secretion signal, TGF targeting domain, a GGGGS (x3), 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.

(21) FIG. 1C shows a chimera containing an OmpA secretion signal, TGF targeting domain, a GGGGS (x3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (A365-380 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.

(22) FIG. 1D shows a chimera containing an OmpA secretion signal, EGF binding peptide (EGB), a GGGGS (x3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (A365-380 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.

(23) FIG. 1E shows a chimera containing an M13 pIII secretion signal, EGF binding peptide (EGB), a GGGGS (x3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (4365-380 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.

(24) FIG. 1F shows a chimera containing an OmpA secretion signal, Her2 affibody binding, a GGGGS SEQ ID NO.: 024 linker, ToxA PE38 (contains Domain II, partial Ib (365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

(25) FIG. 1G shows a chimera containing an OmpA secretion signal, Her2 affibody binding, a GGGGS (x3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (A365-380 amino acids) domain III followed optionally by KDEL retrograde trafficking signal; without KDEL the native signal REDLK SEQ ID NO.: 023.

(26) FIG. 1H shows a chimera containing an OmpA secretion signal, Her2 affibody, a GGGGS (x3) 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.

(27) FIG. 1I shows a chimera containing an OmpA secretion signal, EGFR binding peptide, a GGGGS (x3), 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.

(28) Chimeric Bacterial Toxins

(29) 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).

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

(31) 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.

(32) 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.

(33) 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.

(34) FIG. 2D shows the cldtB, pltB and pltA artificial operon with SexAl, HindIII and Xbal with the FLAG epitope and a GGGGS (x3), SEQ ID NO.: 008 linker inserted in the SexAl and HindIII sites.

(35) FIG. 2E shows the cldtB, pltB and pltA artificial operon with SexAl, HindIII and Xbal with the FLAG epitope and a GGGGS (x3), 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.

(36) Chimeric ToxA Forms Secreted by Bacteria.

(37) 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-GIII-KDEL (Kihara and Pastan 1994, Small Chimeric Toxins Containing Only Transforming Growth Factor and Domain III of Pseudomonas Exotoxin with Good Antitumor Activity in Mice. Cancer Res 1994:54:5154-5159) had a TGF -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 at Different Locations in Pseudomonas Exotoxin. Bioconjugate Chem. 3, 63-68) have no signal peptide, only a single GGGGS SEQ ID NO.: 024 linker with 10 amino acids upstream of DIII, 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.

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

(39) 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.

(40) 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.

(41) FIG. 3C shows the cldtB, pltB and pltA artificial operon with HindIII, Xbal and Clal with the FLAG epitope and a GGGGS (x3), SEQ ID NO.: 008 linker inserted in the Xbal and Clal sites.

(42) FIG. 3D shows the cldtB, pltB and pltA artificial operon with the FLAG epitope and a GGGGS (x3), 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.

(43) Chimeric Cvtolethal Distending Toxins.

(44) 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.

(45) 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.

(46) 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.

(47) Overall improvement is defined as an increase in effect, such as the ability to kill 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.

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

(49) 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.

(50) 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.

(51) FIG. 4C shows inactivation of NLS2 and

(52) 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.

(53) Secreted Protease Inhibitors

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

(55) 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.

(56) 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.

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

(58) 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).

(59) 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 g/mL anhydrotetraclyine HCl after 24 hours at 37 C.

(60) 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).

(61) 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 C.

(62) FIG. 5D shows a staining pattern of protected protein.

(63) 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 2.5103 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.

(64) 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.

(65) 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. The protective effect of YebF-SFTI is shown 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.

(66) FIG. 7A shows cytotoxicity (anticancer cell killing) protease challenge/protease inhibitor assay results, in 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 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.

(67) 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 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.

(68) 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 -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 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.

(69) See also Examples 12-14.

(70) 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.

(71) NT: no treatment.

(72) PBS: phosphate buffered saline.

(73) 1: treatment with 10 L of a bacterial culture supernatant carrying an empty vector plasmid into a 100 L tissue culture wells, with dilutions of 1, 1/2, 1/4, 1/8 and 1/16.

(74) 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.

(75) 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.

(76) 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.

(77) 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.

(78) 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.

(79) 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.

(80) 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.

(81) 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.

(82) 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.

(83) 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.

(84) 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 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 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.

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

(86) FIG. 10A:

(87) NT: no treatment. PBS, phosphate buffered saline.

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

(89) 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.

(90) 2: A chimeric toxin composed of N-terminal TGF 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.

(91) 3: A chimeric toxin composed of N-terminal TGF 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.

(92) 4: A chimeric toxin composed of N-terminal TGF 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.

(93) EV 2: empty vector plasmid.

(94) FIG. 10B:

(95) NT: no treatment. PBS, phosphate buffered saline.

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

(97) 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.

(98) 2: A chimeric toxin composed of N-terminal TGF 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.

(99) 3: A chimeric toxin composed of N-terminal TGF 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.

(100) 4: A chimeric toxin composed of N-terminal TGF 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.

(101) EV 2: empty vector plasmid.

(102) 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.

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

(104) 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 concentration and two-fold dilutions to 1/16 were conducted. The amylase was then added to the mixtures and incubated at 37 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

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

Example 1

(106) A Salmonella expression vector.

(107) 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

(108) Modified TGF-GIII-KDEL (A. Kihara and I. Pastan. Small Chimeric Toxins Containing Only Transforming Growth Factor 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 lb with enhanced activity functionally expressed, secreted, surface displayed and/or released is shown in FIG. 1A. The construct contains an OmpA secretion signal, TGF targeting domain, a GGGGS (x3) 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:

(109) TABLE-US-00001 SEQIDNO.: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

(110) 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:

(111) TABLE-US-00002 SEQIDNO.: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

(112) Modified TGF-GIII-KDEL (A. Kihara and I. Pastan. Small Chimeric Toxins Containing Only Transforming Growth Factor o 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 targeting domain, a GGGGS (x3), 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:

(113) TABLE-US-00003 SEQIDNO.:003 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccg tcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccg cttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaa cgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgatta tttgcacggcgtcacactttgctatgccatagcatttttatccataaga ttagcggatcctacctgacgctttttatcgcaactctctactgtttctc catacccgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGA ATTCACCATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCT GGTTTCGCTACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATT GCCCGGATAGCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCT GGTGCAGGAAGATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGC GCTCGCTGCGAACATGCGGATCTGCTGGCCCATCAACTAGTGGGCGGTG GTGGCAGTGGTGGCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTactgg cgcggagttcctcggcgacggcggcgacgtcagcttcagcacccgcggc acgcagaactggacggtggagcggctgctccaggcgcaccgccaactgg aggagcgcggctatgtgttcgtcggctaccacggcaccttcctcgaagc ggcgcaaagcatcgtcttcggcggggtgcgcgcgcgcagccaggacctc gacgcgatctggcgcggtttctatatcgccggcgatccggcgctggcct acggctacgcccaggaccaggaacccgacgcacgcggccggatccgcaa cggtgccctgctgcgggtctatgtgccgcgctcgagcctgccgggcttc taccgcaccagcctgaccctggccgcgccggaggcggcgggcgaggtcg aacggctgatcggccatccgctgccgctgcgcctggacgccatcaccgg ccccgaggaggaaggcgggcgcctggagaccattctcggctggccgctg gccgagcgcaccgtggtgattccctcggcgatccccaccgacccgcgca acgtcggcggcgacctcgacccgtccagcatccccgacaaggaacaggc gatcagcgccctgccggactacgccagccagcccggcaaaccgccgaaa gatgaactgtaaCTCGAGTagatctgtactCTAGAGTCGACCTGCAGGC ATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATA CAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTG GCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGT GAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA GGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGG GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGA CAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGG GTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAG AAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTG TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCC TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTA AGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGC ACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCT GAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCA ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATT AAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCG TCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGC TTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACC GGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCC CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA AGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCAC ACCGCATATG

(114) 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:

(115) TABLE-US-00004 SEQIDNO.:004 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccg tcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccg cttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaa cgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgatta tttgcacggcgtcacactttgctatgccatagcatttttatccataaga ttagcggatcctacctgacgctttttatcgcaactctctactgtttctc catacccgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGA ATTCACCATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCT GGTTTCGCTACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATT GCCCGGATAGCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCT GGTGCAGGAAGATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGC GCTCGCTGCGAACATGCGGATCTGCTGGCCCATCAACTAGTGGGCGGTG GTGGCAGTGGTGGCGGCGGCTCTGGTGGTGGCGGGTCCCGGGGTctgac ctgcccggtcgccgccggtgaatgcgcgggcccggcggacagcggcgac gccctgctggagcgcaactatcccactggcgcggagttcctcggcgacg gcggcgacgtcagcttcagcacccgcggcacgcagaactggacggtgga gcggctgctccaggcgcaccgccaactggaggagcgcggctatgtgttc gtcggctaccacggcaccttcctcgaagcggcgcaaagcatcgtcttcg gcggggtgcgcgcgcgcagccaggacctcgacgcgatctggcgcggttt ctatatcgccggcgatccggcgctggcctacggctacgcccaggaccag gaacccgacgcacgcggccggatccgcaacggtgccctgctgcgggtct atgtgccgcgctcgagcctgccgggcttctaccgcaccagcctgaccct ggccgcgccggaggcggcgggcgaggtcgaacggctgatcggccatccg ctgccgctgcgcctggacgccatcaccggccccgaggaggaaggcgggc gcctggagaccattctcggctggccgctggccgagcgcaccgtggtgat tccctcggcgatccccaccgacccgcgcaacgtcggcggcgacctcgac ccgtccagcatccccgacaaggaacaggcgatcagcgccctgccggact acgccagccagcccggcaaaccgccgaaagatgaactgtaaCTCGAGTa gatctgtactCTAGAAAGGAGTCGTTATGAAAAAAATAACAGGGATTAT TTTATTGCTTCTTGCAGTCATTATTCTGTCTGCATGTCAGGCAAACTAT ATCCGGGATGTTCAGGGCGGGACCGTATCTCCGTCATCAACAGCTGAAG TGACCGGATTAGCAACGCAGTAACTGCAGGCATGCAAGCTTGGCTGTTT TGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGC AGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGG TCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGA TGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCA AATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGT TGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGG ATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCC GCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGAT GGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACA TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAA TAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCC TTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGA AACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT GTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGC CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG AGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTA TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGG CTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGT TATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAA GTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCT CCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 4

(116) Modified TGF-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 at Different Locations in Pseudomonas Exotoxin. Bioconjugate Chem. 1992, 3, 63-68.)

(117) 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 targeting domain, a GGGGS (x3), SEQ ID NO.: 008 linker, ToxA PE38 (contains Domain II, partial Ib (A365-380 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:

(118) TABLE-US-00005 SEQIDNO.:005 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccg tcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccg cttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaa cgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgatta tttgcacggcgtcacactttgctatgccatagcatttttatccataaga ttagcggatcctacctgacgctttttatcgcaactctctactgtttctc catacccgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGA ATTCACCATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCT GGTTTCGCTACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATT GCCCGGATAGCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCT GGTGCAGGAAGATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGC GCTCGCTGCGAACATGCGGATCTGCTGGCCCATCAACTAGTGGGCGGTG GTGGCAGTGGTGGCGGCGGCTCTGGTGGTGGCGGGTCCCggggtggcag cctggccgcgctgaccgcgcaccaggcttgccacctgccgctggagact ttcacccgtcatcgccagccgcgcggctgggaacaactggagcagtgcg gctatccggtgcagcggctggtcgccctctacctggcggcgcggctgtc gtggaaccaggtcgaccaggtgatccgcaacgccctggccagccccggc agcggcggcgacctgggcgaagcgatccgcgagcagccggagcaggccc gtctggccctgaccctggccgccgccgagagcgagcgcttcgtccggca gggcaccggcaacgacgaggccggcgcggccaacgccccggcggacagc ggcgacgccctgctggagcgcaactatcccactggcgcggagttcctcg gcgacggcggcgacgtcagcttcagcacccgcggcacgcagaactggac ggtggagcggctgctccaggcgcaccgccaactggaggagcgcggctat gtgttcgtcggctaccacggcaccttcctcgaagcggcgcaaagcatcg tcttcggcggggtgcgcgcgcgcagccaggacctcgacgcgatctggcg cggtttctatatcgccggcgatccggcgctggcctacggctacgcccag gaccaggaacccgacgcacgcggccggatccgcaacggtgccctgctgc gggtctatgtgccgcgctcgagcctgccgggcttctaccgcaccagcct gaccctggccgcgccggaggcggcgggcgaggtcgaacggctgatcggc catccgctgccgctgcgcctggacgccatcaccggccccgaggaggaag gcgggcgcctggagaccattctcggctggccgctggccgagcgcaccgt ggtgattccctcggcgatccccaccgacccgcgcaacgtcggcggcgac ctcgacccgtccagcatccccgacaaggaacaggcgatcagcgccctgc cggactacgccagccagcccggcaaaccgccgaaagatgaactgtaaCT CGAGTagatctgtactCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCT GTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGA ACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCG GTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCG CCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGC ATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT CTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGA GCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGAC GCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGAC GGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAA TACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCT TCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT GTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGC CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG AGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTA TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGG CTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGT TATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAA GTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCT CCTTACGCATCTGTGCGGTATTTCACACCGCATATG

(119) 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:

(120) TABLE-US-00006 SEQIDNO.:006 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccg tcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccg cttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaa cgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgatta tttgcacggcgtcacactttgctatgccatagcatttttatccataaga ttagcggatcctacctgacgctttttatcgcaactctctactgtttctc catacccgtttttttgggctagcgaattcgagctCGGTACCCAGGAGGA ATTCACCATGGCTAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCT GGTTTCGCTACCGTAGCGCAGGCAGctGTGGTGAGCCATTTTAACGATT GCCCGGATAGCCATACCCAGTTTTGCTTTCATGGCACCTGCCGCTTTCT GGTGCAGGAAGATAAACCGGCGTGCGTGTGCCATAGCGGTTATGTGGGC GCTCGCTGCGAACATGCGGATCTGCTGGCCCATCAACTAGTGGGCGGTG GTGGCAGTGGTGGCGGCGGCTCTGGTGGTGGCGGGTCCCggggggcagc ctggccgcgctgaccgcgcaccaggcttgccacctgccgctggagactt tcacccgtcatcgccagccgcgcggctgggaacaactggagcagtgcgg ctatccggtgcagcggctggtcgccctctacctggcggcgcggctgtcg tggaaccaggtcgaccaggtgatccgcaacgccctggccagccccggca gcggcggcgacctgggcgaagcgatccgcgagcagccggagcaggcccg tctggccctgaccctggccgccgccgagagcgagcgcttcgtccggcag ggcaccggcaacgacgaggccggcgcggccaacgccccggcggacagcg gcgacgccctgctggagcgcaactatcccactggcgcggagttcctcgg cgacggcggcgacgtcagcttcagcacccgcggcacgcagaactggacg gtggagcggctgctccaggcgcaccgccaactggaggagcgcggctatg tgttcgtcggctaccacggcaccttcctcgaagcggcgcaaagcatcgt cttcggcggggtgcgcgcgcgcagccaggacctcgacgcgatctggcgc ggtttctatatcgccggcgatccggcgctggcctacggctacgcccagg accaggaacccgacgcacgcggccggatccgcaacggtgccctgctgcg ggtctatgtgccgcgctcgagcctgccgggcttctaccgcaccagcctg accctggccgcgccggaggcggcgggcgaggtcgaacggctgatcggcc atccgctgccgctgcgcctggacgccatcaccggccccgaggaggaagg cgggcgcctggagaccattctcggctggccgctggccgagcgcaccgtg gtgattccctcggcgatccccaccgacccgcgcaacgtcggcggcgacc tcgacccgtccagcatccccgacaaggaacaggcgatcagcgccctgcc ggactacgccagccagcccggcaaaccgccgaaagatgaactgtaaCTC GAGTagatctgtactCTAGAAAGGAGTCGTTATGAAAAAAATAACAGGG ATTATTTTATTGCTTCTTGCAGTCATTATTCTGTCTGCATGTCAGGCAA ACTATATCCGGGATGTTCAGGGCGGGACCGTATCTCCGTCATCAACAGC TGAAGTGACCGGATTAGCAACGCAGTAACTGCAGGCATGCAAGCTTGGC TGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAG AACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGC GGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGC GCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGG CATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA TCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGG AGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGA CGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGA CGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAA ATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGC TTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA TGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCC ATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC GAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACT ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAA CAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTG TAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTT ACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG CTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTC TCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 5

(121) An artificial typhoid toxin operon lacking sty and ttsA.

(122) 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:

(123) TABLE-US-00007 SEQIDNO.:007 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccg tcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccg cttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaa cgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgatta tttgcacggcgtcacactttgctatgccatagcatttttatccataaga ttagcggatcctacctgacgctttttatcgcaactctctactgtttctc catacccgtttttttgggctagcgaattcgagctcggtacccaggagga attcaCCATGGTCAAAAAACCTGTTTTTTTCCTTCTGACCATGATCATC TGCAGCTATATTTCTTTTGCCTGCGCTAATATCAGTGACTACAAAGTTA TGACCTGGAATCTTCAGGGCTCTTCAGCATCTACAGAAAGTAAATGGAA TGTCAATGTCAGACAGCTTTTAAGCGGTACTGCCGGTGTGGATATTCTT ATGGTACAGGAGGCCGGTGCTGTTCCCACCTCAGCGGTTCCTACCGGAC GACATATTCAGCCTTTTGGAGTGGGTATTCCCATTGATGAATACACCTG GAATCTCGGAACCACCAGCCGTCAGGATATAAGATATATCTACTACTCG GCTATTGATGTTGGAGCACGCCGTGTTAATCTGGCAATAGTTTCCAGAC AAAGAGCGGATAATGTTTATGTCTTGCGTCCGACAACTGTCGCATCTCG CCCCGTCATTGGCATCGGACTGGGTAATGATGTTTTTCTGACAGCGCAC GCACTGGCTAGTGGAGGTCCGGATGCTGCAGCTATTGTCAGGGTTACCA TTAATTTTTTTAGACAACCTCAGATGCGGCATTTATCCTGGTTTCTTGC CGGGGATTTTAATCGCAGCCCAGACAGACTTGAAAATGACCTGATGACT GAGCATCTGGAACGAGTTGTAGCCGTACTCGCACCTACAGAACCCACGC AAATTAGCGGTGGTATTTTAGATTATGGGGTCATTGTCGATCGAGCACC TTATTCACAAAGGGTCGAAGCATTACGTAATCCACAACTCGCTTCTGAT CATTATCCCGTAGCCTTTTTGGCACGAAGCTGTTAAtgaTCTAGATAAG TCGACgtacAGGAgagtagtATGTATATAAATAAGTTTGTGCCTGTTTA TACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAGTGGACA GGAGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGAATTAC ATGTTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGGTTAA AGCTAACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAGCAG AGCATATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATATT TTTACAGTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCTG GACCTATCCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGA CTATCATCGTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgagggga gaagaaataATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCT TTAATAACTATGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCC GGACGTTATTTTTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAAC TTTCAGCAATTTATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACA GTCGCTATATTGCAACAACCTCAAGTGTTAATCAAACATATGCTATAGC CAGAGCGTACTATTCTCGCTCAACATTCAAAGGTAATTTATACAGATAT CAGATTCGTGCAGATAATAATTTCTACAGCTTGCTCCCATCCATCACCT ATCTGGAAACGCAAGGTGGTCACTTTAATGCTTATGAAAAAACGATGAT GCGATTGCAAAGAGAGTATGTTTCCACATTATCTATTTTACCCGAGAAT ATTCAAAAGGCCGTGGCGCTAGTTTATGATAGCGCAACCGGTCTGGTAA AGGATGGTGTAAGCACAATGAATGCCAGTTATTTAGGTTTAAGCACTAC GTCTAATCCTGGTGTGATACCTTTTCTTCCGGAACCGCAGACGTATACC CAACAACGAATTGATGCATTCGGCCCATTAATAAGTTCATGCTTTTCAA TAGGTAGCGTATGTCAGTCACATCGAGGGCAAAGAGCTGACGTATACAA CATGTCTTTTTATGATGCAAGACCTGTAATAGAACTTATACTTTCTAAA TAAatgaaacttacctatgttgccGCATGCAAGCTAGCTTGGCTGTTTTG GCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGA AGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCC ACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTA GTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAA ACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGT CGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAAC TGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTG CGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAA AGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTT TGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAG TAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTT TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC GTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAG AATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGG CATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACA CTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC GCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC CTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGT TGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTG ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAG TCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT CACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTT TAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGAT CCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT TTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGG TAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGT GTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTA TAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT GCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGC GTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTG ATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAG GAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGG TATTTCACACCGCATATG

Example 6

(124) An artificial typhoid toxin operon lacking sty and ttsA with introduced cloning sites FLAG epitope and GGGGS (x3) SEQ ID NO.: 008 linker at the C-terminus of cldtB.

(125) An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced cloning sites FLAG epitope and GGGGS (x3) 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 (x3) 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 (x3) SEQ ID NO.: 008 from 1194-1238, pltB start at 1278 and stop at 1691 and pltA 1708-2436:

(126) TABLE-US-00008 SEQIDNO.: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

(127) Apoptin C-terminal fusions with typhoid toxin cldtB.

(128) An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced FLAG epitope, GGGGS (x3) 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 to 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:

(129) TABLE-US-00009 SEQIDNO.:010 gggGGCGGCCGCaagaaaccaattgtccatattgcatcagacattgccgtc actgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgctt attaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcg taacaaaagtgtctataatcacggcagaaaagtccacattgattatttgc acggcgtcacactttgctatgccatagcatttttatccataagattagcg gatcctacctgacgctttttatcgcaactctctactgtttctccataccc gtttttttgggctagcgaattcgagctcggtacccaggaggaattcaCCa tgGTCaaaaaacctgtttttttccttctgaccatgatcatctgcagctat atttcttttgcctgcgctaatatcagtgactacaaagttatgacctggaa tcttcagggctcttcagcatctacagaaagtaaatggaatgtcaatgtca gacagcttttaagcggtactgccggtgtggatattcttatggtacaggag gccggtgctgttcccacctcagcggttcctaccggacgacatattcagcc ttttggagtgggtattcccattgatgaatacacctggaatctcggaacca ccagccgtcaggatataagatatatctactactcggctattgatgttgga gcacgccgtgttaatctggcaatagtttccagacaaagagcggataatgt ttatgtcttgcgtccgacaactgtcgcatctcgccccgtcattggcatcg gactgggtaatgatgtttttctgacagcgcacgcactggctagtggaggt ccggatgctgcagctattgtcagggttaccattaatttttttagacaacc tcagatgcggcatttatcctggtttcttgccggggattttaatcgcagcc cagacagacttgaaaatgacctgatgactgagcatctggaacgagttgta gccgtactcgcacctacagaacccacgcaaattagcggtggtattttaga ttatggggtcattgtcgatcgagcaccttattcacaaagggtcgaagcat tacgtaatccacaactcgcttctgatcattatcccgtagcctttttggca cgaagctgtaTAccaggtgattataaagatgacgatgacaaaggcggtgg cggtagcggcggtggcggttctggcggtggcggtagtaagcttATGGCAT ACGGTCGCAAGAAACGTCGTCAACGCCGCCGCATGAACGCCCTGCAAGAA GACACGCCGCCGGGTCCGAGCACGGTTTTTCGTCCGCCGACCAGCTCTCG CCCGCTGGAAACGCCGCATTGCCGTGAAATTCGCATCGGCATTGCAGGTA TCACCATTACGCTGTCTCTGTGCGGCTGTGCAAACGCACGTGCACCGACC CTGCGCTCCGCTACGGCGGATAACAGTGAATCCACCGGTTTTAAAAATGT GCCGGATCTGCGTACGGACCAGCCGAAGCCGCCGTCAAAAAAGCGTTCGT GTGACCCGAGCGAATATCGCGTTAGCGAACTGAAAGAATCTCTGATTACC ACGACCCCGTCCCGTCCGCGCACCGCaaaACGCCGCATCCGTCTGTCTAG ATAGgtcgacgtacAGGAgagtagtATGTATATAAATAAGTTTGTGCCTG TTTATACATTATTAATTCTCATTTATTCTTTTAATGCCAGCGCTGAGTGG ACAGGAGATAATACGAACGCCTATTACTCAGACGAAGTTATCAGTGAATT ACATGTTGGTCAGATAGATACTAGTCCTTATTTTTGCATAAAAACGGTTA AAGCTAACGGTAGTGGTACACCAGTTGTTGCATGTGCGGTATCAAAGCAG AGCATATGGGCGCCCTCCTTTAAAGAACTTCTTGATCAGGCAAGATATTT TTACAGTACAGGGCAATCCGTAAGGATTCATGTTCAAAAAAATATCTGGA CCTATCCGCTTTTTGTTAATACCTTTTCAGCAAATGCTCTTGTGGGACTA TCATCGTGCAGTGCGACACAATGCTTTGGACCCAAGTAAgaggggagaag aaataATGAAAAAGTTAATATTCTTAACCTTATCTATAGTTAGCTTTAAT AACTATGCTGTAGATTTTGTGTATCGTGTGGACTCAACCCCGCCGGACGT TATTTTTCGCGATGGGTTTTCACTACTTGGGTATAACCGTAACTTTCAGC AATTTATTAGTGGAAGGTCATGTAGTGGTGGAAGTAGTGACAGTCGCTAT ATTGCAACAACCTCAAGTGTTAATCAAACATATGCTATAGCCAGAGCGTA CTATTCTCGCTCAACATTCAAAGGTAATTTATACAGATATCAGATTCGTG CAGATAATAATTTCTACAGCTTGCTCCCATCCATCACCTATCTGGAAACG CAAGGTGGTCACTTTAATGCTTATGAAAAAACGATGATGCGATTGCAAAG AGAGTATGTTTCCACATTATCTATTTTACCCGAGAATATTCAAAAGGCCG TGGCGCTAGTTTATGATAGCGCAACCGGTCTGGTAAAGGATGGTGTAAGC ACAATGAATGCCAGTTATTTAGGTTTAAGCACTACGTCTAATCCTGGTGT GATACCTTTTCTTCCGGAACCGCAGACGTATACCCAACAACGAATTGATG CATTCGGCCCATTAATAAGTTCATGCTTTTCAATAGGTAGCGTATGTCAG TCACATCGAGGGCAAAGAGCTGACGTATACAACATGTCTTTTTATGATGC AAGACCTGTAATAGAACTTATACTTTCTAAATAAatgaaacttacctatg ttgccGCATGCAAGCTAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTT CAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGA ATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAAC TCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGC GAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAG TAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCG GAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAG CAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTT TGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTT AAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACT CACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC ATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAAC GTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAAC AATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA GCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTC ATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCT GCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC GCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTT CGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATAC CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGC GGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTT GCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG GATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCG AACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGA TGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

Example 8

(130) N-terminal fusions with typhoid toxin cldtB.

(131) An artificial, inducible typhoid toxin operon lacking sty and ttsA and containing introduced FLAG epitope, GGGGS (x3) 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 paratyphi A Ncol_F1: 5-TACGCCATGGTCAAAAAACCTGTTTTTTTCCTTCTGACC-3 SEQ ID NO.: 011 and HindIII_Xbal Clal-R1: 5-CGATCCCTCTAGACCCAAGCTTGTCACTGATATTAGCGCAGGC-3 SEQ ID NO.: 012 (set 1) and HindIII_Xbal_Clal-F1: 5-GCTTGGGTCTAGAGGGATCGATTACAAAGTTATGACCTGGAATCTTCAG-3 SEQ ID NO.: 013 and cldtB_stop_Xbal_Sall-R1: 5-GATCGTCGACTTATCTAGATCATTAACAGCTTCGTGCCA-3 SEQ ID NO.: 014 (set 2).

(132) 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).

(133) TABLE-US-00010 SEQIDNO.: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

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

(135) 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 (x3), 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).

(136) TABLE-US-00011 SEQIDNO.: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

(137) 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

(138) 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 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

(139) 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

(140) 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:

(141) TABLE-US-00012 SEQIDNO.: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

(142) 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:

(143) TABLE-US-00013 SEQIDNO.: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

(144) 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:

(145) TABLE-US-00014 SEQIDNO.:019 CATATGGGGGCGGCCGCCTAAGACCCACTTTCACATTTAAGTIGTTTTTC 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

(146) 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.