Process for producing recombinant proteins using a gene for tRNA
AssigneeRoche Diagnostics GmbH
InventorsUlrich Brinkmann, Ralf Mattes, Stephan Fischer
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Abstract
The present invention provides 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 recognises the codons AGG and AGA is increased to at least fivefold of the amount normally occurring in these cells.
Claims10
A method for increasing production of a protein which contains arginine, comprising the steps of:
- (i) transforming an Escherichia coli host cell with an expression vector which contains a DNA sequence, wherein said DNA sequence (a) codes for the arginine containing protein, and (b) contains codons AGG and AGA;
- (ii) transforming said Escherichia coli host cell with an extrachromosomal expression vector which contains a DNA sequence which codes for a tRNA, wherein said tRNA (a) incorporates arginine into protein, (b) recognizes both of codons AGA and AGG, and (c) contains anticodon UCU, and
- (iii) treating the transformed host cell resulting from step (ii) under conditions favoring an increase in expression of said DNA sequence which codes for said tRNA, so as to produce the arginine containing protein in an amount greater than the amount of the arginine containing protein that could be produced by the transformed host cell prior to step (ii).
Plasmid pUBS 98.sky1 (DSM 4898).
A method for enhanced expression of a gene which codes for an arginine containing protein, comprising:
- transforming an Escherichia coli host cell with:
- a first vector which contains (i) a gene which codes for tRNA which (a) incorporates arginine into a protein, (b) recognizes both codon AGA and codon AGG and (c) contains the anticodon UCU, and (ii) a first ori;
- and
- a second vector which contains (i) a DNA sequence which (a) codes for the arginine containing protein and (b) contains AGA and AGG codons, and (ii) a second ori different from said first ori, and culturing the transformed Escherichia coli host cell under conditions favoring an increase in the expression of said gene which codes for said tRNA, so as to produce the arginine containing protein in an amount greater than the amount of the arginine containing protein that could be produced by an E. coli host cell which is not transformed with said first vector, wherein said tRNA incorporates arginine into the arginine containing protein.
Dependencies are inferred from the claim text.