大肠杆菌F0F1 - ATP酶γ和ε亚基复合物的体内形成、纯化及结晶

Formation in vivo, purification and crystallization of a complex of the gamma and epsilon subunits of the F0F1-ATPase of Escherichia coli.

作者信息

Cox G B, Cromer B A, Guss J M, Harvey I, Jeffrey P D, Solomon R G, Webb D C

机构信息

Department of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra City, A.C.T.

出版信息

J Mol Biol. 1993 Feb 20;229(4):1159-62. doi: 10.1006/jmbi.1993.1113.

DOI:10.1006/jmbi.1993.1113

PMID:8445643

Abstract

A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads. The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography. Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant. The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A. The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit.

摘要

一种由大肠杆菌F1 - ATP酶（ECF1 - ATP酶）的ε亚基和谷胱甘肽 - S - 转移酶γ亚基（ECF1 - ATP酶的）融合蛋白组成的复合物在体内形成，并通过与谷胱甘肽 - 琼脂糖珠结合从细胞提取物中纯化出来。通过凝血酶消化从复合物中释放出谷胱甘肽 - S - 转移酶，并通过阳离子交换色谱法纯化γ/ε复合物。使用PEG8000作为沉淀剂，通过气相扩散法生长复合物的晶体。晶体为正交晶系，空间群为P2(1)2(1)2，a = 161.9 Å，b = 44.1 Å，c = 63.4 Å。不对称单元的体积与一个γ亚基和一个ε亚基的复合物的存在一致。