Xu Junjie, Song Jian, Yan Fei, Chu Huiying, Luo Jixun, Zhao Yongshan, Cheng Xu, Luo Guimin, Zheng Qingchuan, Wei Jingyan
College of Pharmaceutical Science, Jilin University, Changchun, PR China.
J Mol Recognit. 2009 Jul-Aug;22(4):293-300. doi: 10.1002/jmr.946.
Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.
谷胱甘肽过氧化物酶（GPX）是抗氧化酶家族的重要成员之一。它可以催化谷胱甘肽还原氢过氧化物，以保护细胞免受氧化损伤。在先前的研究中，我们通过化学突变将催化基团硒代半胱氨酸（Sec）引入结合位点，制备了具有GPX活性的人催化抗体Se-scFv-B3（克隆B3的含硒单链Fv片段）；然而，其活性并不十分令人满意。为了尝试提高其GPX活性，我们对scFv-B3进行了结构分析。通过同源建模构建了scFv-B3的三维（3D）结构，并进行了结合位点分析。还对抗体-谷胱甘肽（GSH）复合物进行了计算机辅助对接和能量最小化（EM）计算。从这些模拟中，候选结合位点中的Ala44和Ala180分别被选择突变为丝氨酸，随后可将其转化为催化性Sec基团。这两种突变蛋白和scFv的野生型均在大肠杆菌Rosetta中以可溶形式表达，并通过镍（2+）固定金属亲和色谱（IMAC）纯化，然后通过对活性丝氨酸残基进行化学修饰转化为具有GPX活性的含硒催化抗体。与原始的Se-scFv-B3相比，突变催化抗体Se-scFv-B3-A180S的GPX活性显著提高。
