| PATENT ASSIGNEE'S COUNTRY | Canada |
| UPDATE | 12.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 07.12.99 |
| PATENT TITLE |
Expression of gene products from genetically manipulated strains of bordetella |
| PATENT ABSTRACT |
An expression system for expressing gene products from recombinant Bordetella strains and specific nucleic acid molecules useful in transforming Bordetella strains for such expression are described. A nucleic acid molecule may comprise a Bordetella promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product with the heterologous gene transcriptionally regulated by the Bordetella promoter. The nucleic acid molecule may further comprise a further nucleic acid molecule encoding a leader sequence for secretion of the non-Bordetella gene product. Another nucleic acid molecule may comprise a Bordetella promoter coupled to a nucleic acid sequence encoding a non-Bordetella leader sequence for secretion of a gene product, which may be a Bordetella gene product or a non-Bordetella gene product. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 26.01.98 |
| PATENT REFERENCES CITED |
Johnson and Burns. 1994. J. Bacterial. 176: 5350. Locht et al. 1992. Embo J. 11: 3175. Zealey et al. 1992. Appl. Environ. Microbiol. 58: 208. Burnette. 1994. Structure 2: 151. Holmgren et al. 1993. Vaccine 11: 1179. Wu and Russell. 1994. Vaccine 12: 215. Dertzbough and Elson. 1993. Infect. Immun. 61: 384. Cardenas and Clement. 1993. Vaccine 11: 126. Airaksinen et al. 1991. Biotechnology Lett 13: 305. Lebens et al. 1993. Bio/Technology 11: 1574. Burnette et al. 1991. Infect. Immun. 59: 4266. Klauser et al. 1990. Embo J. 1991. Nicosia et al. 1986. Proc. Natl. Acad. Sci. (USA) 83: 4631. Loosmore et al. 1989. Nucleic Acid. Res. 17: 8365. Relman et al. 1989. Proc. Natl. Acad. Sci. (USA) 86: 2637. Charles et al. 1989. Proc. Natl. Acad. Sci. (USA) 86: 3554. Dams et al. 1991. Biochen. Biophys. Acta. 1090: 139. Zealey et al. 1990. Bio/Technology 8: 1025. Imaizumi et al. 1983. Infect. Immun. 41: 1138. Nicosia et al. 1987. Infect. Immun. 55: 963. Makoff et al. 1990. Bio/Technology 8: 1030. Domenighimi et al. 1990. Molec. Microbiol. 4: 787. Watson, M. 1984. Nucl. Acids. Res. 12: 5145. Charles et al. 1989 Proc. Natl. Acad Sci 86 3554-3558. Gross et al. 1992 Res. Microbiol. 143:671-681. Scarlato et al. 1991 Mol. Microbiol. 5:2493-2498. Li et al. 1991. Mol. Microbiol. 5:409-417. Stibitz et al. 1986. Gene 50:133-140. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What we claim is: 1. A nucleic acid molecule comprising a Bordetella promoter which is the fha promoter of Bordetella pertussis operatively coupled to a heterologous gene encoding a non-Bordetella gene product, wherein the heterologous gene is transcriptionally regulated by the Bordetella promoter, and further comprising a nucleic acid sequence encoding a leader sequence of a Bordetella protein which is the leader sequence of the PRN protein of Bordetella pertussis for the secretion of the non-Bordetella gene product. 2. The nucleic acid molecule of claim 1 wherein the heterologous gene encodes a cholera toxin molecule. 3. The nucleic acid molecule of claim 2 wherein the cholera toxin molecule is the B subunit thereof. 4. The nucleic acid molecule of claim 1 further comprising a first DNA sequence corresponding to a 5'-flanking sequence of fha gene and disposed at the 5'-end of the nucleic acid molecule and a second DNA sequence corresponding to a 3'-flanking sequence of fha gene and disposed at the 3'-end of the nucleic acid molecule, said first and second sequences permitting specific integration of the nucleic acid molecule into a Bordetella genome at the fha locus. 5. The nucleic acid molecule of claim 4 wherein said Bordetella is a strain of B. pertussis and said 3' and 5' flanking regions are the 3' and 5' flanking sequences of the fha gene of B. pertussis. 6. A plasmid adapted for transformation of a Bordetella strain comprising the nucleic acid molecule of claim 1. 7. The plasmid of claim 6 which is JB-1141-5. 8. A recombinant strain of Bordetella containing the nucleic acid molecule of claim 1 integrated into the genome thereof and secreting said non-Bordetella gene product. 9. The strain of claim 8 which is a strain of B. pertussis. 10. A method of expression of a non-Bordetella gene product, which comprises culturing the recombinant strain of claim 8 and isolating the resulting gene product. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD TO THE INVENTION The present invention relates to the field of molecular biology and is particularly concerned with the expression of gene products from strains of Bordetella. BACKGROUND OF THE INVENTION Bordetella pertussis, the organism responsible for whooping cough, expresses a number of virulence factors, such as pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN). These proteins are secreted by the organism through the use of signal peptides and/or accessory genes (refs. 1 and 2--Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). We have previously demonstrated that it is possible to manipulate the expression of these Bordetella proteins through alteration of gene copy number (ref. 3) or the use of hybrid genes with autologous promoters (ref. 4). For example, the amount of secreted and processed PT holotoxin was increased more than 3-fold by increasing the copy number of the tox operon encoding PT (ref. 5). The amount of secreted and processed pertactin was increased 8-fold by using a hybrid gene which replaced the native prn promoter with the stronger fha promoter. The yield of pertactin was further increased to 20-fold wild-type levels by adding a second copy of the hybrid gene. Many gene products including proteins and polypeptides of commercial and medical significance are only available in small amounts from their natural sources, are difficult to isolate or require modification of, for example, their primary amino acid sequence for optional use and/or activity. Thus, many genes have been expressed by recombinant DNA means in a variety of microbial hosts, including bacterial hosts. The gene expressed in the microbial host is typically heterologous to the host. Examples of bacterial hosts used for expression of heterologous proteins include strains of Escherichia coli, Salmonella species (ref. 10) and Bacillus subtilis (ref. 11). Particular biological properties of strains of Bordetella make them attractive hosts for the production of certain heterologous gene products. Thus, many of the antigens produced by B. pertussis are large, can be multimetric and may require post-translational assembly or processing. For example, the pertactin antigen is produced as a 93-kDa precursor and the mature protein is produced by excision of the N-terminal signal peptide and removal of a C-terminal fragment. Pertussis toxin is a 105 kDa exotoxin produced by B. pertussis, and is encoded by the TOX operon and consists of five polypeptide subunits (S1 to S5) arranged in the typical A-B structure of bacterial toxins. The S2, S3, S4 and S5 subunit form a pentamer (the B oligomer) which, when combined with the S1 subunit forms the holotoxin. For PT, for example, such complex assembly cannot be achieved in E. coli (ref. 22) and, for the 69 kd material, protein accumulated as insoluble inclusion bodies in E. coli (ref. 23). This intracellular expression in E. coli is to be contrasted with the secretion of soluble antigens by B. pertussis strains. FHA is another large molecule (Mwt 220 kDa) secreted by B. pertussis (ref. 24). Vibrio cholerae is the organism that causes cholera, a severe disease of dehydration caused by diarrhoea. Many of the symptoms of cholera can be attributed to the action of cholera toxin (CT), which like B. pertussis PT, is an A/B toxin with ADP-ribosyl transferase activity. However, unlike PT which has four different B subunit components comprising a pentamer, CT has a pentameric structure made up of identical subunits (ref. 6) Cholera toxin has been shown to have considerable use as a mucosal adjuvant and the B subunit alone may be sufficient to generate a mucosal response in some instances (ref. 7). A response is generated if cholera toxin B (CTB) is either co-administered or chemically coupled to another protein (ref. 8). Chimeric genes have also been engineered which have foreign epitopes fused to cholera toxin B and the resultant fusion proteins can sometimes induce an immune response to the foreign epitope (ref. 9). Cholera toxin B has been expressed from recombinant V. cholerae (ref. 12), E. coli (ref. 13), and S. typhimurium (ref. 14). Although B. pertussis has been used to over-express autologous proteins by gene manipulation (refs. 4 and 5), it has not heretofore been used to produce heterologous proteins. SUMMARY OF THE INVENTION The present invention is directed towards recombinant strains of Bordetella which express non-Bordetella gene products. Accordingly, in one aspect of the present invention, there is provided a nucleic acid molecule comprising a Bordetella promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product, wherein the heterologous gene is transcriptionally regulated by the-Bordetella promoter. The non-Bordetella gene product may be one of a wide variety of proteins and polypeptides. The protein or peptide may be an enzyme, an enzyme inhibitor, an antigen, an immunogen, an allergen, a hormone, a lymphokine, an immunoglobulin or fragment thereof, a toxin, a toxin subunit, a mammalian protein, a structural protein or a receptor. The invention is illustrated by the expression of a cholera toxin molecule as the non-Bordetella gene product, specifically the B subunit of cholera toxin. However, any other protein or polypeptide may comprise the expressed non-Bordetella gene product. The Bordetella promoter employed in the nucleic acid molecule provided in accordance with this aspect of the invention may be any of the Bordetella promoters, preferably the tox, prn and fha promoters from any Bordetella strain, including B. pertussis. The heterologous gene component of the nucleic acid molecule provided in accordance with this aspect of the invention may further comprise a nucleic acid sequence encoding a leader sequence for secretion of the non-Bordetella gene product. The leader sequence may be any sequence mediating secretion of the non-Bordetella gene product. In one embodiment, the leader sequence is a leader sequence of a Bordetella protein or subunit thereof or a fragment or analog of the Bordetella protein leader sequence retaining secretion-mediating properties. The leader sequence may be the Bordetella pertactin leader sequence or a pertussis toxin subunit leader sequence, such as that for the S1 subunit, of any Bordetella strain, including B. pertussis. Alternatively, in another embodiment, the leader sequence is a leader sequence of a non-Bordetella protein or subunit thereof or a fragment or analog of the non-Bordetella protein leader sequence retaining secretion-mediating properties. The non-Bordetella gene product may be a secreted gene product, in which case the non-Bordetella leader sequence preferably is the native leader sequence of the secreted gene product. In an illustrative example of the latter embodiment of the invention, the secreted gene product may be a cholera toxin molecule, for example, the B subunit thereof and the leader sequence may be the cholera toxin. B subunit leader sequence. Specific combinations of promoter, leader sequence and heterologous gene product sequence are provided herein, including those designed toxp/CTB-L/ctb, fhap/CTB-L/ctb, toxp/S1-L/ctb, toxp/PRN-L/ctb, fhap/S1-L/ctb and fha/PRN-L/ctb. The nucleic acid molecule provided in accordance with this aspect of the invention may further comprise a first DNA sequence corresponding to a 5' flanking sequence of a selected Bordetella gene and disposed at the 5' end of the nucleic acid molecule and a second DNA sequence corresponding to a 3' flanking sequence of the selected Bordetella gene and disposed at the 3' end of the nucleic acid molecule. The first and second DNA sequences permit specific integration of the nucleic acid molecule into the genome of a Bordetella species, preferably B. pertussis, at a locus corresponding to the selected Bordetella gene. The Bordetella promoter present in the nucleic acid molecule may be that of the selected Bordetella gene providing the flanking sequences. The selected Bordetella gene may be any of the Bordetella genes, including the tox, prn or fha gene of a Bordetella strain, preferably B. pertussis. The nucleic acid molecule with flanking regions as described above may be provided in a plasmid adapted for transformation of a Bordetella strain, preferably a B. pertussis strain. Specific plasmids have been prepared herein, as described in more detail below and are identified as plasmids DS-546-1, JB-898-2-1, DS-729-1-1, DS-729-2-1, JB-1201-4 and JB-1141-5. Another aspect of the invention provides a recombinant strain of Bordetella, which may be a B. pertussis strain, a B. parapertussis strain, a B. bronchiseptica strain or a B. avium strain, particularly a B. pertussis strain, containing the nucleic acid molecule provided in the above-described aspect of the invention, integrated into the genome thereof and expressing the non-Bordetella gene product. One specific recombinant B. pertussis strain provided herein is B. pertussis strain 694-46, which has been deposited with the American Type Culture Collection, Rockville, Md., U.S.A., on Jan. 11, 1995 under the terms of the Budapest Treaty as ATCC Accession number 55,654. The non-Bordetella gene product may be obtained by culturing a recombinant strain provided herein. In a further aspect of the invention, there is provided a nucleic acid molecule comprising a Bordetella promoter coupled to a nucleic acid sequence encoding a non-Bordetella leader sequence for secretion of a gene product. The secreted gene product may be a Bordetella gene product or a non-Bordetella gene product. The Bordetella promoter component of the nucleic acid molecule provided in accordance with this further aspect of the invention may be any of the Bordetella promoters, preferably the tox, prn or fha promoter of a Bordetella strain, preferably B. pertussis. The non-Bordetella leader sequence component of the nucleic acid molecule provided in accordance with this further aspect of the invention may be any of the leader sequences mediating secretion of a gene product, including bacterial (prokaryotic) leader sequences (such as E. coli leader sequences including rlpB, pal ompA, pilin gene leader sequences and H. influenzae leader sequences including the transferrin receptor protein leader sequence), eukaryotic leader sequences (including mammalian) and viral leader sequences. Some appropriate leader sequences for use in aspects of the present invention are described in reference 25. In an illustrative example of this further aspect of the invention, the non-Bordetella leader sequence may be that for the cholera toxin B subunit. A recombinant strain of Bordetella, such as a B. pertussis strain, a B. parapertussis strain, B. bronchiseptica strain or a B. avium strain, preferably a B. pertussis strain, may contain the nucleic acid molecule provided in accordance with this further aspect of the invention and secrete the gene product. Such recombinant strain of Bordetella may be cultured under a range of appropriate conditions to secrete the gene product. In an additional aspect of the invention, the heterologous gene may be provided with optimized codons for Bordetella expression. One example of such heterologous gene has the nucleic acid sequence of FIG. 1, which encodes the B-subunit of cholera toxin and constitutes an embodiment of this additional aspect of the invention. The present invention, therefore, provides an expression system for expressing gene products from recombinant Bordetella strains and specific nucleic acid molecules useful in transforming Bordetella strains for such expression. The expressed gene products have a variety of uses, depending on the form and nature of the product produced, as will be evident to a person skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further understood from the following detailed description and examples with reference to the accompanying drawings, in which: FIG. 1 shows the sequence of a synthetic cholera toxin B subunit gene (SEQ ID NO: 1) and its derived amino acid sequence (SEQ ID NO: 2), based upon strain 569B of V. cholerae; FIG. 2 shows the construction scheme for plasmid DS-546-1 which contains the toxp/CTB-L/ctb gene; FIG. 3 shows the sequences of oligonucleotides (SEQ ID NOS: 3 to 16) used for the construction of plasmid DS-546-1; FIG. 4 shows the construction scheme for plasmid JB-898-2-1 which contains the fha/CTB-L/ctb gene; FIG. 5 shows the sequences of oligonucleotides (SEQ ID NOS: 17 to 20) used to construct plasmid JB-898-2-1; FIG. 6 shows the construction scheme for plasmid DS-729-1-1 which contains the toxp/S1-L/ctb gene; FIG. 7 shows the sequences of oligonucleotides (SEQ ID NOS: 21 to 30) used' to construct plasmid DS-729-1-1; FIG. 8 shows the construction scheme for plasmid DS-729-2-1 which contains the toxp/PRN-L/ctb gene; FIG. 9 shows the sequences of oligonucleotides (SEQ ID NOS: 31 to 40) used to construct plasmid DS-729-2-1; FIG. 10 shows the construction scheme for plasmid JB-1201-4 which contains the fhap/S1-L/ctb gene; FIG. 11 shows the sequences of oligonucleotides (SEQ ID NOS: 41 to 48) used to construct plasmid JB-1201-4; FIG. 12 shows the construction scheme for plasmid JB-1141-5 which contains the fhap/PRN-L/ctb gene; FIG. 13 shows the sequences of oligonucleotides (SEQ ID NOS: 49 to 56) used to construct plasmid JB-1141-5; FIG. 14 shows the chromosomal maps of the genes at the fha and tox loci and the corresponding Southern blot showing the correct chromosomal integration. Chromosomal DNA was digested with Bgl II and hybridized with the approximately 300 bp ctb probe indicated in the figure. Lane 1, strain 694-46 (fhap/PRN-L/ctb); lane 2, strain 694-54 (fha/S1-L/ctb); lane 3, strain 694-12 (toxp/PRN-L/ctb); lane 4, strain 694-4 (toxp/S1-L/ctb); lane 5, strain 10536 (wild-type B. pertussis); and FIG. 15 shows the SDS PAGE and corresponding Western blot of recombinant B. pertussis strain 694-46 which expresses CTB. Lane 1, acetone precipitated supernatant from strain 694-46 (fha/PRN-L/ctb), boiled in SDS; lane 2, acetone precipitated supernatant from strain 694-46, unboiled; lane 3, cell pellet from strain 694-46, boiled in SDS; lane 4, cell pellet from strain 694-46, unboiled; lane 5, acetone precipitated supernatant-from strain 10536 (wild-type B. pertussis); lane 6, cell pellet from strain 10536; lane 7, purified cholera toxin (Sigma), boiled in SDS; lane 8, purified cholera toxin, unboiled. In some of the above figures, the following abbreviations are used: toxp is the B. pertussis tox promoter fhap is the B. pertussis fha promoter ctb is the synthetic cholera toxin B gene (SEQ ID NO:1) CTB-L is the sequence encoding the cholera toxin B subunit leader sequence S1-L is the sequence encoding the pertussis toxin subunit S1 leader sequence PRN-L is the sequence encoding the pertussis pertactin leader sequence Restriction enzyme recognition sites are B, BamH I; Bg, Bgl II; H, Hind III; K, Kpn I; R, EcoR I; S, Sac I; and Hf, HinfI CAP is calf alkaline phosphatase GENERAL DESCRIPTION OF-THE INVENTION Bordetella pertussis 10536 is the vaccine production strain of the assignee hereof and it has been used as the initial strain for all the work detailed by the inventors herein. The genes for B. pertussis PT, fha and pertactin and V. cholerae CT have been cloned and sequenced (Refs. 15 to 19) and the promoter regions and transcriptional starts of the structural genes have been determined. The inventors have generated hybrid genes by substituting the B. pertussis native structural genes encoding a leader sequence and/or mature protein by gene segments encoding native, autologous, or heterologous leader peptides and a mature foreign protein. This was accomplished by fusing the promoters with the gene segment encoding the leader peptide at the ATG start codon, followed by the structural gene for the mature foreign protein joined at the natural cleavage site of the signal sequence. Such fusions result in a native promoter, a native, autologous, or heterologous leader peptide, and a heterologous structural gene. The resultant hybrid genes then have been integrated by homologous recombination into the chromosome of B. pertussis at the loci corresponding to the gene from which the promoters were derived. As examples of the use of hybrid genes expressing foreign proteins, genes have been created containing a tox promoter with the cholera toxin B leader peptide and mature cholera toxin B sequence; a tox promoter with the pertussis toxin subunit S1 leader peptide and mature cholera toxin B sequence; a tox promoter with the pertactin leader peptide and mature cholera toxin B sequence; an fha promoter with the cholera toxin B leader peptide and mature cholera toxin B sequence; an fha promoter with the pertussis toxin subunit S1 leader peptide and mature cholera toxin B sequence; and an fha promoter with the pertactin leader peptide and mature cholera toxin B sequence. A number of B. pertussis strains have been generated to demonstrate the success of this strategy. The efficiency of expression of the foreign CTB protein is dependent upon both the promoter and the leader peptide which precede the structural ctb gene. The use of the fha promoter in the hybrid genes results in a higher level of expression of the foreign protein than when the tox promoter is used. This phenomenon was also observed when hybrid genes were used to express autologous proteins from B. pertussis (ref. 4). For the leader peptides, expression levels varied as follows: pertactin>PT subunit S1>cholera toxin B. The best combination of promoter and leader peptide in the hybrid genes expressing foreign proteins was the fha promoter with the pertactin leader peptide. The CTB expressed by the recombinant B. pertussis strains is produced and is a pentamer as demonstrated by SDS PAGE and Western blot analysis of unboiled (pentameric) and boiled (monomeric) samples. The CTB binds to GM1 as demonstrated by ELISA. Thus, a complex foreign protein which has authentic structure and binding functions can be secreted by the recombinant Bordetella strains of the invention. It has been clearly demonstrated that the structural gene of a foreign protein may be fused to a B. pertussis promoter through a gene fragment encoding a native, autologous, or heterologous leader peptide to express foreign proteins. BIOLOGICAL DEPOSITS B. pertussis strain 694-46 which contains the fha/PRN-L/ctb hybrid gene at the fha locus has been deposited with the American Type Culture Collection (ATCC) located at Rockville, Md., U.S.A., pursuant to the Budapeast Treaty and prior to the filing of this application. The ATCC access number is 55,654. Samples of the deposited strain will become available to the public upon the grant of a patent based on this United States patent application. The invention described and claimed herein is not limited in scope by the strain deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar strains to that deposited are within the scope of the invention. |
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