Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA.
An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.
Widersten
M
M
Department of Biochemistry, Uppsala University, Sweden.
Huang
M
M
Mannervik
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002847:Chromatography, Agarose; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D013862:Thiogalactosides / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013862:Thiogalactosides; D005982:Glutathione Transferase