Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-12-01
1999-08-24
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
536 231, 536 241, 4353201, 4352523, C12P 2106, C07H 1700, C12N 1500
Patent
active
059424184
DESCRIPTION:
BRIEF SUMMARY
FIELD OF 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 Bordetalla 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 multimeric 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. FRA 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 to
REFERENCES:
patent: 5395764 (1995-03-01), Riboli et al.
Dams et al. 1991 Biochemica et Biophysica Acta 1090.:139-141.
Gross et al. 1992 Res Microbiol 143:671-681.
Li et al. 1991 Mol. Microbiol. 5:409-417.
Scarlato et al 1991 Mol. Microbiol. 5:2493-2498.
Stibitz et al. 1986 Gene 50:133-140.
Klein Michel H.
Loosmore Sheena M.
Yacoob Reza K.
Zealey Gavin R.
Carlson Karen Cochrane
Connaught Laboratories Limited
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