Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli.
Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme. Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein. The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region. The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E. coli. Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency. The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening. Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression. Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E. coli.
Johansson
A S
AS
Department of Biochemistry, Biomedical Center, Uppsala University, Uppsala, S-751 23, Sweden.
Bolton-Grob
R
R
Mannervik
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
D018388:Codon, Terminator; D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase