Improving GPX activity of selenium-containing human single-chain Fv antibody by site-directed mutation based on the structural analysis.
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.
Xu
Junjie
J
College of Pharmaceutical Science, Jilin University, Changchun, PR China.
Song
Jian
J
Yan
Fei
F
Chu
Huiying
H
Luo
Jixun
J
Zhao
Yongshan
Y
Cheng
Xu
X
Luo
Guimin
G
Zheng
Qingchuan
Q
Wei
Jingyan
J
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002999:Clone Cells; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary; D012643:Selenium / Q000378:metabolism*; D017279:Selenocysteine / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
D017151:Antibodies, Catalytic; D007135:Immunoglobulin Variable Region; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D005978:Glutathione; D012643:Selenium