Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA.
An expression plasmid, pKK-GTB2, containing the complete coding sequence of a rat liver glutathione S-transferase Yc subunit was constructed and expressed in Escherichia coli. The entire Yc cDNA sequence from plasmid pGTB42 (Telakowski-Hopskins et al., 1985, J. Biol. Chem. 260, 5820-5825) was amplified by the polymerase chain reaction, subcloned into modified expression vector A6316 (Schoner et al., 1986, Proc. Natl. Acad. Sci. USA 83, 8506-8510 and Linemeyer et al., 1987, Bio/Technology 5, 960-965) and transformed into E. coli strain AB1899. The colonies were screened by hybridization to pGTB42 and the production of Yc subunit was detected by immunoblot analysis. The purified recombinant Yc subunit was active in the conjugation and peroxidation reactions, and appeared homogeneous as judged by sodium dodecyl sulfate gel electrophoresis. Amino acid sequencing of the expressed Yc subunit revealed that about 40% of the expressed protein was blocked at the N-terminus. Approximately 25% of the sequenceable protein (15% of total protein) contained the initiation methionine residue at the amino terminus whereas the rest of the sequenceable protein had proline as the N-terminus. In contrast, only one molecular species with Pro as the first amino acid was identified when the inducer isopropyl-beta-D-thiogalactopyranoside was omitted in the growth medium. Our observation indicated that under certain growth conditions, the enzymes responsible for protein maturation were not able to complete the processing of the overproduced recombinant Yc in E. coli.
Department of Animal and Exploratory Drug Metabolism, Merck Sharp & Dohme Research Labortories, Rahway, New Jersey 07065.
S E
SE
Huskey
R W
RW
Wang
D L
DL
Linemeyer
C B
CB
Pickett
A Y
AY
Lu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000096:biosynthesis; D004274:DNA, Recombinant / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity
D004274:DNA, Recombinant; D004247:DNA; D005982:Glutathione Transferase