Human catalytic antibodies with glutathione peroxidase activity

Citation
F. Fang et al., Human catalytic antibodies with glutathione peroxidase activity, J INORG BIO, 85(4), 2001, pp. 301-307
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics","Inorganic & Nuclear Chemistry
Journal title
JOURNAL OF INORGANIC BIOCHEMISTRY
ISSN journal
0162-0134 → ACNP
Volume
85
Issue
4
Year of publication
2001
Pages
301 - 307
Database
ISI
SICI code
0162-0134(200107)85:4<301:HCAWGP>2.0.ZU;2-J
Abstract
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 h ighly homologous, consisting of 786 bps and belonging to the same VH family -DP25. In the premise of maintaining the amino acid sequence, mutated plasm ids were constructed by use of the mutated primers in PCR, and they were ov er-expressed in E. coli. After the active site serine was converted into se lenocysteine with the chemical modifying method, we obtained two human cata lytic antibodies with GPx activity of 72.2U/mu mol and 28.8U/mu mol, respec tively. With the aid of computer mimicking, it can be assumed that the anti bodies 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. (C) 2001 Elsevier Science BY. All rights reserved.