title: [Cloning and characterization of lactate dehydrogenase C4 from pika ochotona curzoniae].
abstract: 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 RACE method. The cDNA was 1498 bp in length containing 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 degrees 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 microM 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.
authors.forename: Q -L
authors.initials: Q-
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Yang
authors.forename: Y -Y
authors.initials: Y-
authors.lastname: Zhao
authors.forename: S -Z
authors.initials: S-
authors.lastname: Zhang
authors.forename: Q -H
authors.initials: Q-
authors.lastname: He
authors.forename: X -Y
authors.initials: X-
authors.lastname: Meng
authors.forename: W -R
authors.initials: W-
authors.lastname: Tan
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007793:Lagomorpha / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; C409673:lactate dehydrogenase C4