Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity.
In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol.
Huo
Rui
R
College of Pharmaceutical Science, Jilin University, Changchun 130021, P.R. China.
Wei
Jingyan
J
Xu
Junjie
J
Lv
Shaowu
S
Zheng
Qingchuan
Q
Yan
Fei
F
Su
Jiaming
J
Fan
Jia
J
Li
Jieshuai
J
Duan
Yujing
Y
Yu
Yang
Y
Jin
Fenghai
F
Sun
Weiguo
W
Shi
Yi
Y
Cong
Dengli
D
Li
Wei
W
Yan
Ganglin
G
Luo
Guimin
G
D017151:Antibodies, Catalytic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D004353:Drug Evaluation, Preclinical; D005978:Glutathione / Q000031:analogs & derivatives / Q000276:immunology; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D019151:Peptide Library; D017279:Selenocysteine / Q000737:chemistry
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D005978:Glutathione