Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli.
A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259, 5182-5188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using MaeI endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tac promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Bal31 to remove extra nucleotides from the 5' noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cytosol is apparently blocked.
Wang
R W
RW
Department of Animal & Exploratory Drug Metabolism, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
Pickett
C B
CB
Lu
A Y
AY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004247:DNA; D005982:Glutathione Transferase