Chu Ching-Hsuen, Cheng Dong
Department of Obesity and Metabolic Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, NJ 08543-5400, USA.
Protein Expr Purif. 2007 Jun;53(2):421-7. doi: 10.1016/j.pep.2007.01.012. Epub 2007 Feb 2.
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.
本研究报道了利用杆状病毒系统表达具有功能活性的人重组3-甲基巴豆酰辅酶A羧化酶（MCCC），该酶是一种异源多聚体复合物，由分别由不同基因编码的α和β亚基组成。通过免疫亲和纯化，已开发出一种有效的方案来纯化活性MCCC，在Superose-6凝胶过滤色谱中，活性MCCC似乎存在于约500-800kDa的复合物中。与天然酶一致，在重组人MCCC中，α和β亚基的化学计量比为1:1。重组酶的k(cat)值测定为约4.0s(-1)。它还具有与报道的天然酶相似的K(m)值（ATP：45±11μM；3-甲基巴豆酰辅酶A：74±7μM）。本文所述的重组人MCCC可为测试针对旨在治疗肥胖、2型糖尿病和其他代谢紊乱的乙酰辅酶A羧化酶抑制剂的交叉反应性提供一种反筛酶源。
