Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-03-31
1997-08-12
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352535, 4353201, C12P 2106, C12N 1563, C12N 1576
Patent
active
056564538
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a process for the production of recombinant proteins in streptomycetes in which the expression is carried out in Streptomyces galbus DSM 40480.
Streptomycetes are used on a large scale for the industrial production of various proteins. A review of 821 of these strains is given by P. Kampfer et al. (J. of General Microbiology 137 (1991), 1831-1891).
Processes for the large-scale fermentation of streptomycetes have therefore been developed to an advanced stage and the use of streptomycetes in the production of therapeutic products has already been proven several times. Streptomycetes are also suitable for use in the recombinant production of proteins. The transformation processes necessary for this have already been described for many different streptomycetes strains (R. Hatter et al., in Actinomycetes in Biotechnology, eds. Goodfellow, Williams and Mordaski, Academic Press, London, 1988, 89-148; see in particular Table 10, p. 141-145). One problem with the expression of heterologous DNA in streptomycetes is, however, the cleavage of heterologous DNA, e.g. from E. coli, in genetically well characterized streptomycetes strains, such as for example Streptomyces coelicolor. The expression of heterologous proteins in Streptomyces galbus and Streptomyces lividans is described by A. Weib et al. (6th German VAAM Workshop on the Biology of Streptomyces, Sept. 30-Oct. 3, 1990). Streptomyces lividans is preferred for the expression of heterologous proteins in streptomycetes since it shows a low cleavage activity towards heterologous DNA (T. Kieser et al. Meth. Enzymology 204 (1991), 430-458). The amount of protein which is obtained after expression in Streptomyces lividans is, however, unsatisfactory for large-scale production.
The object of the invention is therefore to provide a process for the production of recombinant proteins in streptomycetes in which the recombinant protein is obtained in a high yield.
This object is achieved by a process for the production of recombinant proteins in streptomycetes cells in which the cells are transformed with a vector that contains a DNA sequence coding for the protein, the transformed cells are multiplied such that the protein is expressed and the protein is isolated from the cells or the fermentation supernatant. This process is characterized in that the expression is carried out in Streptomyces galbus DSM 40480.
It surprisingly turned out that a substantially higher yield of recombinant protein is achieved when using Streptomyces galbus DSM 40480 than when using Streptomyces lividans. Thus serine protease B from Streptomyces griseus is obtained in a 10-50-fold higher yield after cloning and expression in Streptomyces galbus DSM 40480 than after cloning and expression in Streptomyces lividans under otherwise identical conditions (see FIG. 2).
Competent protoplasts of Streptomyces galbus DSM 40480 are preferably produced for the transformation by lysozyme treatment and freezing at -70.degree. C. Transformation of these competent protoplasts with the vector DNA can be carried out in the presence of polyethylene glycol (PEG). After the transformation an incubation for 30 hours at 20.degree.-25.degree. C. is carried out in order to regenerate the protoplasts before selecting transformed streptomycetes. In order to express the heterologous DNA, the streptomycetes are then cultured in a suitable complex or synthetic medium. The selection of suitable media known to a person skilled in the art essentially depends on the heterologous protein to be produced and thus on the purification steps to be used. The recombinant protein is then isolated from the cells or from the fermentation supernatant according to known processes.
An expression vector is preferably used for the production of the recombinant protein in which the DNA sequence coding for the recombinant protein is under the control of a strong promoter. The ermE promoter (M. Bibb et al., Gene 41 (1986) 357-368) of the erythromycin resistance gene (ermE) from Saccharopolyspora erythreus is particularly pre
REFERENCES:
patent: 5242809 (1993-09-01), Adams et al.
Kampfer et al., J. Gen. Microbiol., 137:1831-1891 (1991).
Kieser et al., Meth. Enzymol., 204:430-458 (1991).
Hutter et al., Actinomycetes in Biotechnology, Academic Press, London, 89-148 (1988).
Henderson et al., J. Bacteriol., 169:3778-3784 (1987).
Hopwood et al., Genetic Manipulation of Streptomyces, A laboratory Manual, John Innes Foundation, Norwich (1985).
Chater et al., Current Topics Microbiol., 46:69-95 (1982).
Bibb, M.J., Janssen, G.R., and Ward, J.M. 1985. Cloning and Analysis of the Promoter Region of the Erythromycin-Resistance Gene (ermE) of Streptomyces erythraeus. Gene, 38(1-3):215-226.
Piepersberg Wolfgang
Rossler Carola
Weiss Angela
Boehringer Mannheim GmbH
Robinson Douglas W.
Wai Thanda
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