High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.
A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.
Kolm
R H
RH
Department of Biochemistry, Uppsala University, Sweden.
Stenberg
G
G
Widersten
M
M
Mannervik
B
B
D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D007527:Isoenzymes; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase