Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC) |
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Authors: | Chu Ching-Hsuen Cheng Dong |
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Institution: | Department of Obesity and Metabolic Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, NJ 08543-5400, USA. |
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Abstract: | 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. |
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Keywords: | 3-Methylcrotonyl-CoA carboxylase Stoichiometry Recombinant expression |
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