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.
Yang
Y F
YF
Department of Biochemistry, Michigan State University, East Lansing 48824.
Wells
W W
WW
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