Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1988-02-26
1992-09-01
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352523, 4353201, 536 27, 935 48, C12N 1562, C12N 1563, C12N 1509, C07H 2104
Patent
active
051438305
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to recombinant DNA biotechnology and in particular to processes for the production of polypeptide products in transformed host cells wherein the polypeptide products are secreted from the host cells.
BACKGROUND TO THE INVENTION
In recent years advances in recombinant DNA biotechnology have made it possible to produce a wide variety of useful polypeptide products in host cells which have been transformed and transfected with DNA sequences which code for production of the polypeptide products. Thus hormones such as insulin and growth hormones e.g. human growth hormone, and industrially or therapeutically useful enzymes, such as chymosin and tissue plasminogen activator (tPA) have been produced in transformed host cells.
Bacterial cells, in particular E. coli, have been used as host cells for the production of recombinant polypeptide products. The genetic systems of such bacterial cells are now relatively well understood and also such cells exhibit good growth characteristics. However, when such bacterial cells are used to overproduce foreign proteins, the foreign products typically accumulate within the host cells and it is usually necessary to disrupt the cells to effect recovery of the products. Also recombinant products are often produced within bacterial host cells in the form of insoluble aggregates in which the polypeptides are not in their native, biologically functional form. It is necessary, therefore, to solubilise and denature/renature the insoluble polypeptide products to obtain useful products in soluble, native, biologically functional form. For instance, British Patent No. GB 2100732B describes inter alia processes for production of methionine-prochymosin in E. coli involving disruption of the bacterial host cells and treatment with urea or guanidine HCl to solubilise the unnatural prochymosin-containing aggregates which are produced. The processes of cell disruption and denaturation/renaturation add significantly to the cost of producing recombinant polypeptide products.
Attempts have been made therefore to develop bacterial expression systems which secrete recombinant products into the extracellular culture medium. For example, recombinant heterologous polypeptides have been expressed in bacteria as fusion proteins in which the heterologous polypeptide sequence is joined with an N-terminal signal sequence. However, such fusion proteins, although exported across the inner membrane in Gram-negative bacteria with concomitant removal of the signal sequence, fail to cross the outer membrane and remain within the periplasm. Thus, it is still necessary to disrupt the host cells to effect recovery of heterologous recombinant products and denaturation/renaturation treatment may be required to yield products in native, biologically functional form.
Also `leaky` mutants of Gram-negative bacterial host cells such as E. coli have been proposed for use in the production and secretion of products to the extracellular medium. However, such mutant cells are often not suitable for large scale production of heterologous protein products since the yield of product is generally low and the fragility of the cells makes them unsuitable for growing on a large scale.
Haemolysin (Hly) is an extracellular protein toxin which is produced by some strains of E. coli, and as such is one of the few proteins produced by Gram-negative bacteria which are transported across both the cytoplasmic and outer membranes. Hitherto, however, the processes by which haemolysin is secreted to the extracellular medium have not been satisfactorily explained. Studies have indicated that a specific transport system determined by two of the hly genes is responsible for transport of haemolysin across the outer membrane (Wagner et al, J. Bacteriol. 154, 200, 1983), and at least four genes the hly A, hly B, hly C and hly D genes, are required to elicit a cell-free haemolytic phenotype. The hly C gene product appears to be required for activation of the hly A gene product which provides the haemol
REFERENCES:
Felmlee et al., Jul. 1985 J. Bact. 163(1) 94-105.
Oliver D. B. et al., Jan. 1985, J. Bact. 161(1) 285-291.
Holland Ian B.
Mackman Nigel
Nicaud Jean-Marc
Schwartz Richard A.
Ulm John D.
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