Abstract
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 Ni2+-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/μmol.
Original language | English (US) |
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Pages (from-to) | 324-329 |
Number of pages | 6 |
Journal | Journal of Molecular Recognition |
Volume | 21 |
Issue number | 5 |
DOIs | |
State | Published - 2008 |
Externally published | Yes |
Keywords
- Antibody library
- Catalytic antibody
- Chemical modification
- Glutathione peroxidase (gpx)
- Selenium
- Single-chain fv fragment (scfv)
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology