Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.