Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1990-10-10
1992-06-16
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4353201, C12N 1579, C12N 1569
Patent
active
051224640
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to recombinant DNA sequences, vectors containing them, and a method for the use thereof. In particular, the present invention relates to recombinant DNA sequences which encode the complete amino acid sequence of a glutamine synthetase (GS) (L-glutamate:ammonia ligase [ADP-forming]; EC 6.3.1.2) and to the use of such nucleotide sequences.
The ability of cloned genes to function when introduced into host cell cultures has proved to be invaluable in studies of gene expression. It has also provided a means of obtaining large quantities of proteins which are otherwise scarce or which are completely novel products of gene manipulation. It is advantageous to obtain such proteins from mammalian cells since such proteins are generally correctly folded, appropriately modified and completely functional, often in marked contrast to those proteins as expressed in bacterial cells.
Where large amounts of product are required, it is necessary to identify cell clones in which the vector sequences are retained during cell proliferation. Such stable vector maintenance can be achieved either by use of a viral replicon or as a consequence of integration of the vector into the host cell's DNA.
Where the vector has been integrated into the host cell's DNA, the copy number of the vector DNA, and concomitantly the amount of product which could be expressed, can be increased by selecting for cell lines in which the vector sequences have been amplified after integration into the host cell's DNA.
A known method for carrying out such a selection procedure is to transform a host cell with a vector comprising a DNA sequence which encodes an enzyme which is inhibited by a known drug. The vector may also comprise a DNA sequence which encodes a desired protein. Alternatively the host cell may be co-transformed with a second vector which comprises the DNA sequence which encodes the desired protein.
The transformed or co-transformed host cells are then cultured in increasing concentrations of the known drug hereby selecting drug-resistant cells. It has been found that one common mechanism leading to the appearance of mutant cells which can survive in the increased concentrations of the otherwise toxic drug is the over-production of the enzyme which is inhibited by the drug. This most commonly results from increased levels of its particular mRNA, which in turn is frequently caused by amplification of vector DNA and hence gene copies.
It has also been found that, where drug resistance is caused by an increase in copy number of the vector DNA encoding the inhibitable enzyme, there is a concomitant increase in the copy number of the vector DNA encoding the desired protein in the host cell's DNA. There is thus an increased level of production of the desired protein.
The most commonly used system for such co-amplification uses as the enzyme which can be inhibited dihydrofolate reductase (DHFR). This can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, a host cell which lacks an active gene which encodes DHFR is either transformed with a vector which comprises DNA sequences encoding DHFR and a desired protein or co-transformed with a vector comprising a DNA sequence encoding DHFR and a vector comprising a DNA sequence encoding the desired protein. The transformed or co-transformed host cells are cultured in media containing increasing levels of MTX, and those cell lines which survive are selected.
Other systems for producing co-amplification have been employed. However, none of them has been as widely used as the DHFR/MTX system.
The co-amplification systems which are at present available suffer from a number of disadvantages. For instance, it is generally necessary to use a host cell which lacks an active gene encoding the enzyme which can be inhibited. This tends to limit the number of cell lines which can be used with any particular co-amplification system. For instance, there is at present only one cell line known which lacks the gene encoding DHFR. It would be advantageous if an effective
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Bebbington Christopher R.
Wilson Richard H.
Celltech Limited, a British Company
Nolan S. L.
Schwartz Richard A.
The University Court of the University of Glasgow
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