Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli.
The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.
Bücheler
U S
US
Institut für Biochemie II, Universität Heidelberg, F.R.G.
Werner
D
D
Schirmer
R H
RH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005980:Glutathione Reductase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D004247:DNA; D005980:Glutathione Reductase