Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli.
The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.
LeVan
K M
KM
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208.
Goldberg
E
E
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013737:Testis / Q000201:enzymology*
D007527:Isoenzymes; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase