Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1986-03-18
1990-05-15
Teskin, Robin
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 435255, 435256, 435320, 4351721, 4352402, 935 28, 935 37, 935 56, 935 69, 536 27, C12P 2100, C12P 2132, C12N 1500, C12N 500
Patent
active
049257919
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the field of recombinant DNA biotechnology. In particular the invention relates to a eukaryotic expression vector, a host organism transformed with the eukaryotic expression vector, specific DNA sequences and to a process for the production of polypeptide.
BACKGROUND TO THE INVENTION
In recent years, advances in biotechnology have made possible the production of desirable polypeptide products by inserting an appropriate heterologous gene into a host organism and subsequently culturing the organism to produce the polypeptide. In broad terms, these techniques involve the insertion of a structural gene coding for the desired polypeptide into a vector capable of stable existence in the cells of a host organism. The gene is inserted into the vector in a position relative to appropriate expression control sequences such that once within the host organism, the vector expresses the inserted gene to produce the polypeptide. Such vectors are referred to in the art as "expression vectors" and have been the subject of considerable research. The main thrust of the research has been to develop expression vectors which are compatible with prokaryotic host organisms such as bacteria (for example, Escherichia coli (E. coli)), and eukaryotic host organisms such as yeasts (for example, Saccharomyces cerevisiae) and cells of higher organisms (for example mammalian cells in tissue culture). A wide variety of polypeptides have been produced, such as animal and human hormones, enzymes and other useful proteins. The research has involved detailed studies of expression control sequences affecting expression, and in particular, promoter sequences, which are responsible for directing transcription of genetic material.
The commercial use of expression systems is hampered by the lethal or debilitating effect of toxic expression products upon the host organism. It has been recognised therefore that it is desirable to regulate heterologous gene expression. In a regulated expression system, the host organism can be cultured to produce a high cell density whilst expression of a gene in an inserted vector is kept at a low level. When the host organism reaches an appropriate cell density, expression may be induced, for example, by adding a material having a regulating effect on the culture medium. A large number of expression control sequences (including promoters) which allow a degree of expression regulation have been identified both for prokaryotic and eukaryotic host organisms. In published European Patent Application EP-A2-0073635, a yeast expression vector is described which makes use of the control sequences of the yeast phosphoglycerate kinase (PGK) gene and which allows expression level control by adjusting the level of fermentable carbon in the culture medium. Published European patent application EP-A2-0118393 describes a prokaryotic and eukryotic expression system based upon the control sequences of heat-shock genes derived from Drosophlia melanogaster. The control sequences include temperature-dependent promoters which allow for expression level control by adjusting the temperature of the culture medium. British patent specification No. 1557774 and published International patent application No. WO 84/01171 describe prokaryotic expression level control systems in which the average number of plasmids in each cell (the copy number) is controlled. Copy number control allows a regulation of the net gene expression occurring in each cell of the host organism.
The known regulated expression systems for eukaryotic host organisms do not have the ability to control expression over a wide range of expression levels. In many cases, it is not possible to reduce the concentration of toxic products, by promoter control alone, to a level at which the cell growth is unaffected and yet to allow for a significant production of the desired polypeptide when required.
The object of the present invention is to provide a eukaryotic expression vector capable of controlling the expression level of a h
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Miller Allan M.
Nasmyth Kim A.
Celltech Limited
Teskin Robin
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