Lepesheva G I, Usanov S A
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Zhodinskaya 5/2, Minsk, 220141 Belarus.
Biochemistry (Mosc). 1998 Feb;63(2):224-34.
Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc. The quantitative values of the studied parameters are practically identical in natural and substrate-bound recombinant cytochrome P450scc, while there were great differences between substrate-bound and substrate-free forms of recombinant cytochrome P450scc both of functional (decrease of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in the reconstituted system, and affinity to adrenodoxin for substrate-free cytochrome P450scc) as well as structural (increase in accessibility to exogenous and endogenous proteolysis) character. The identity of the folding process for recombinant and natural proteins as well as the nature of a stabilizing and activating effect of cholesterol on cytochrome P450scc is discussed.
对重组细胞色素P450scc在大肠杆菌中进行异源表达条件的优化后，每升细菌培养物中细胞色素P450scc的表达水平约为420纳摩尔。本文描述了一种以底物结合的高自旋形式和无底物的低自旋形式纯化重组蛋白的新方法。高度纯化的电泳纯重组细胞色素P450scc，无底物形式和底物结合形式每毫克蛋白分别含有12.3和16.7纳摩尔血红素。对来自牛肾上腺皮质线粒体的重组和天然细胞色素P450scc进行了功能和免疫化学比较。测定了细胞色素P450scc与胆固醇和肾上腺皮质铁氧还蛋白复合物的解离常数、重组系统（NADPH - 肾上腺皮质铁氧还蛋白还原酶 - 肾上腺皮质铁氧还蛋白）中的酶促还原效率以及胆固醇侧链裂解活性。发现用胰蛋白酶对重组细胞色素P450scc进行有限的蛋白水解会形成两个主要片段，它们在电泳和免疫化学上与牛肾上腺皮质细胞色素P450scc蛋白水解过程中形成的片段F1（29.8 kD）和F2（26.6 kD）相同。在天然和底物结合的重组细胞色素P450scc中，所研究参数的定量值实际上是相同的，而重组细胞色素P450scc的底物结合形式和无底物形式在功能（胆固醇侧链裂解活性降低、重组系统中的酶促还原效率以及无底物细胞色素P450scc对肾上腺皮质铁氧还蛋白的亲和力）和结构（对外源和内源蛋白水解的可及性增加）特征方面都存在很大差异。本文讨论了重组蛋白和天然蛋白折叠过程的一致性以及胆固醇对细胞色素P450scc的稳定和激活作用的性质。
