High-level expression of pig liver thioltransferase (glutaredoxin) in Escherichia coli.
We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.
Department of Biochemistry, Michigan State University, East Lansing 48824.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D054477:Glutaredoxins; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009154:Mutation; D010088:Oxidoreductases; D010957:Plasmids; D011506:Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013552:Swine; D014169:Transformation, Bacterial
D000596:Amino Acids; D054477:Glutaredoxins; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA; D010088:Oxidoreductases