Human catalytic antibodies with glutathione peroxidase activity.
In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied.
Fang
F
F
Lab 5, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China.
Wang
L
L
Qi
X J
XJ
Yan
X Y
XY
Ding
L
L
Zhao
D Q
DQ
Ni
J Z
JZ
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data
D017151:Antibodies, Catalytic; D004247:DNA; D005979:Glutathione Peroxidase