Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1993-01-27
2001-08-07
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071200, C435S320100, C435S375000
Reexamination Certificate
active
06270988
ABSTRACT:
The present invention is concerned with a process for the expression of a recombinant gene which contains AGA and/or AGG codons for arginine in
Escherichia coli
after transformation with an expression vector which contains the recombinant gene.
The gene-technological production of proteins or protein-containing gene products is one of the main objects of modern biotechnology. Since, in particular, prokaryotes are suitable for the production of comparatively large amounts of protein because of their easy fermentability, attempts have already been made many times to express eukaryotic genes in prokaryotes. However, difficulties arise since eukaryotic proteins are often only produced in small amounts in prokaryote cells and the cells show fermentation/growth difficulties in the case of increased production of the proteins.
Such problems are observed, inter alia, in the case expression of the tissue type plasminogen activator t-PA. However, the production of this protein is an object worth striving for since, in clinical trials, it has proved to be suitable for the treatment of infarct diseases. Although
E. coli
cells express t-PA cDNA introduced on a plasmid or vector in a satisfactory amount, nevertheless the cells weaken in the case of the formation of biomass during the fermentation so that, in all, the production of t-PA does not reach the state which could be expected when starting from the production of a single cell. The plasmid stability in the
E. coli
cells is also low and, due to loss of the coding plasmid, the rate of production decreases further. Therefore, hitherto it has only been possible to achieve an expression of about 5% of the total cell protein as t-PA (Rothstein and Bertonis, Gene, 61, 41-50/1987).
It is an object of the invention to provide a process for enhanced yield of expression of gene products. The process is superior to known processes where notably poorer growth of host cells, as well as plasmid instability was observed. Both factors led to low yields of gene product.
Thus, according to the present invention, there is provided a process for the expression of a recombinant gene, which contains AGA and/or AGG codons for arginine, in
Escherichia coli
after transformation with an expression vector which contains the recombinant gene, wherein the amount of t-RNA present in the
E. coli
cells which incorporates arginine and recognizes the codons AGG and AGA is increased to at least fivefold of the amount normally occurring in these cells.
In the scope of the present invention, by the term t-RNA which recognizes AGA/AGG codons and incorporates arginine, there is to be understood not only the corresponding t-RNA occurring naturally in
E. coli
but also those synthetic, mutated or suppressor t-RNA's which display these properties.
The process according to the present invention makes it possible to produce, in distinctly increased amounts, recombinant genes whose expression in the past led to only low yields of the desired proteins because of poor growth of the
E. coli
host cells. This result is unexpected because, when there is a shortage of t-RNA, protein synthesis would be expected to be low due to poor expression of the foreign gene. In fact, the foreign gene is well expressed, but the host cell suffers.
In a preferred embodiment of the present invention, the amount of t-RNA which recognises the AGA/AGG codon and incorporates arginine, hereinafter simply referred to as t-RNA, is increased in the
E. coli
cells by introducing into the cells at least one gene coding for such t-RNA. This can preferably take place by introducing one or more extra-chromosomal expression vector into the cell.
The natural t-RNA which incorporates arginine into
E. coli
and recognizes the codons AGA and AGG is a product of the dnaY gene which was described by Garcia et al. in Cell, 45, 453-459/1986. The whole sequence of this gene is known: it contains 118 essential base pairs and is preferably used for increasing the amount of t-RNA in
E. coli
by introduction of the gene. However, it is also possible to introduce into the cells a gene for a synthetic, mutated or suppressor t-RNA which incorporates arginine and recognizes the AGA/AGG codon. It is also possible to use the gene of the t-RNA of the phage T
4
.
In a preferred embodiment of the present invention, the t-RNA gene or genes, together with the eukaryotic gene, are introduced on the same expression vector into the cell. When the expression vector is a high copy plasmid, there is thereby already provided a sufficient increase of the intracellular t-RNA level so that no negative effects are to be observed on the host cells in the case of the expression of the eukaryotic gene.
In another preferred embodiment, the t-RNA gene and the eukaryotic gene are introduced on different expression vectors into the
E. coli
cells. The positive effect of making available a t-RNA Arg(AGG/AGA) during the expression of recombinant protein in
E. coli
is, not dependent upon the fact that the gene for this t-RNA is present in the cis-position on the expression vector in question. This t-RNA can also be coded on a second vector (trans-active). However, this additional vector must be compatible with the expression vector in the
E. coli
cell, i.e. have a replication origin different from that of the expression plasmid.
It is possible to use a vector with a smaller plasmid copy number than the expression plasmid but also, by the use of a vector which is present in higher copy number than the expression vector on which the eukaryotic gene is present, to achieve a still higher t-RNA level in comparison with the eukaryotic gene. In the case of the introduction of the t-RNA gene and of the eukaryotic gene on the same expression vector, it is not only possible to use two genes under the control of different promoters but also both genes in the form of an operon under the control of the same promotor. The only thing that is important is that a stop codon is present between the eukaryotic gene and the t-RNA gene so that no fusion protein is obtained following transcription.
In another preferred embodiment of the present invention, the t-RNA gene or genes is or are incorporated into the chromosome of the host cell. It is especially preferred to integrate the gene in such a manner that it is expressed under a strong promotor. For this purpose, the t-RNA gene or genes is or are preferably introduced simultaneously, together with a strong promotor under the control of which the t-RNA is expressed.
Known methods are used for the introduction of a t-RNA gene into the chromosome, as well as for the introduction of the gene into an expression vector, as well as for the transformation of the host cells. For the introduction into the chromosome, methods which use transposons or phages, and use recombination phenomena are preferred
A further preferred possibility of increasing the amount of t-RNA in the cell is to change the natural promotor of the chromosomal gene which codes for this t-RNA in such a way that the t-RNA is formed in larger amounts. For this purpose, it is especially preferred to exchange the whole natural promotor for a stronger, preferably inducable and/or repressable promotor. Such promotors are known to the expert, as well as the processes for the exchange of the promotors which again are preferably carried out with the use of transposons or phages.
The present invention also provides a process for the expression of a recombinant gene which contains AGA and/or AGG codons for arginine in
E. coli
by transformation with an expression vector which contains the recombinant gene, wherein an
E. coli
strain is selected which contains at least the fivefold amount of the amount of t-RNA normally occurring in
E. coli
which recognizes the codons AGG and AGA and incorporates arginine, this strain being used as host cell.
Previously known strains mostly have only a small amount of t-RNA which incorporates arginine and which recognises the codons AGA/AGG and there occur the results described hereinbefore in the case of the express
Brinkmann Ulrich
Fischer Stephan
Mattes Ralf
Fulbright & Jaworski LLP
McKelvey Terry
Roche Diagnostics GmbH
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