Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1987-12-15
1991-01-29
Moskowitz, Margaret
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
435 6952, 4351723, 4353201, 424 857, C12N 1521, C12N 121, A61K 3766
Patent
active
049886223
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the art of genetic engineering and biotechnology and, more particularly, to a novel in vitro engineered recombinant plasmid DNA pVN 22 coding the biosynthesis of human leukocyte interferon .alpha.-I1, and to a novel strain Pseudomonas sp. 31 (pVN 22) producing human leukocyte interferon .alpha.-I1 containing this plasmid.
PRIOR ART
Interferon--proteins produced by certain types of cells in response to viral infections, as well as in response to the effect of some other agents. Interferons exhibit an antiviral effect and a number of other properties: inhibition of cell growth, influence on their differentiation, activation of macrophages, increasing the number of antibody-independent killer cells, and the like. Interferons produced by human leukocytes are referred to as leukocyte interferons (interferons .alpha.) and comprise a family of related proteins containing 166 (in one case 165) aminoacid moieties. The homology of the aminoacid sequences for different interferons .alpha. is as high as 80% and over. Every kind of interferon .alpha. is coded by its own gene and after induction of the synthesis of interferons by a corresponding agent a mixture of interferons .alpha. is delivered into the blood plasma. The biological activity of different interferons .alpha. determined by their ability of protecting cells from the effect of viruses considerably varies; their other biological and physico-chemical properties are also different. In the preparation of interferons .alpha. for pharmaceutical purposes donor's blood is conventionally employed. However, this source is not capable of fully meeting the demand in high-purity interferons. The use of bacterial producers-strains created by methods of molecular cloning and genetic engineering enables preparation of interferons .alpha. in substantially any required amounts. However, every individual producer gives only one kind of interferon .alpha. out of a great number thereof. To obtain a pharmaceutical preparation having properties close to those of a natural compound, it is necessary to be in possession of a whole range of producers synthesizing various kinds of interferons .alpha. (e.g., .alpha.-A, .alpha.-B, .alpha.-C, .alpha.-1 and the like), so that a high-purity interferons .alpha. could be further intermixed in such proportions that usually prevail in the human blood. Sufficiently active bacterial producers of some subtypes of interferons .alpha. have already been prepared by methods of the genetic engineering. Thus, engineered were recombinant plasmids ensuring biosynthesis of interferons .alpha.-A, .alpha.-F, .alpha.-K and others in cells of E. coli. However, this is not sufficient for obtaining preparations fully reproducing the effect of a mixture of interferons .alpha. obtained from the donor's blood.
Known in the art is a recombinant plasmid DNA pLeIF-rL synthesized on the basis of the vector pBR 322 and coding the biosynthesis of interferon .alpha.-L; also known is the strain Escherichia coli 294 containing this plasmid (EPA2 0072541). The gene of interferon .alpha.-L is prepared from the library of human genes using the phague .lambda. Haron 4A. In the plasmid pLeIF-rL the synthesis of interferon .alpha.-L is controlled by an inducible tryptophane promotor.
This strain is characterized by that during its fermentation on enriched media with a relatively high content of tryptophane (LB-medium, Hottinger's broth) usually employed for ensuring a rapid growth of bacteria, a low level of biosynthesis of the desired product is observed. This disadvantage is connected with the fact that the synthesis of interferon .alpha.-L is effected under control of the regulatory area of the tryptophane promotor the operation of which must be induced. To obtain a high level of biosynthesis of interferon .alpha.-L, the cultural broth should be diluted with a minimal salt medium by 20-40 times. A considerable decrease of tryptophane concentration in the medium results in normalization of transcription of the gene a
REFERENCES:
patent: 4680260 (1987-07-01), Dehahov et al.
patent: 4748233 (1988-05-01), Sloma
Nature vol. 287, 2 Oct. 1980.
Chistoserdov Andrei J.
Dolganov Grigory M.
Eremashvili Marina R.
Evdonina Ljudmila V.
Jurin Vitaly L.
Guest Shelly J.
Moskowitz Margaret
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