Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

• Published in: Biochemical journal 1991-02, Vol.273 (3), p.587-592
• Main Authors: LEVAN, K. M, GOLDBERG, E
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Chromatography, Affinity; Cloning, Molecular - methods; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Isoenzymes; Kinetics; L-Lactate Dehydrogenase - genetics; L-Lactate Dehydrogenase - isolation & purification; L-Lactate Dehydrogenase - metabolism; Male; Molecular Sequence Data; Open Reading Frames; Oxidoreductases; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Restriction Mapping; testes; Testis - enzymology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2730587

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.