Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC).
The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a approximately 500-800kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of alpha and beta subunits are at a one:one ratio. The k(cat) value of the recombinant enzyme is determined to be approximately 4.0s(-1). It also possesses K(m) values (ATP: 45+/-11microM; 3-methylcrotonyl-CoA: 74+/-7microM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders.
Chu
Ching-Hsuen
CH
Department of Obesity and Metabolic Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, NJ 08543-5400, USA.
Cheng
Dong
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D019735:Carbon-Carbon Ligases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase