Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240–1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251–255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5–10%, 15–20% and 25–30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8M urea by Co2+-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46×105M−1 and 1.03×105M−1 respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394units/μmol, which approaches the activity of rabbit liver GPX.
- artificial enzyme
- chemical mutation
- enzyme mimics
- protein engineering
Abbreviations used: 2F3-scFv, single-chain Fv fragment of monoclonal antibody 2F3; GPX, glutathione peroxidase; GSHPx-1, classical cellular GPX; IMAC, immobilized metal-affinity chromatography; IPTG, isopropyl β-d-thiogalactoside; N2ph, 2,4-dinitrophenyl; scFv, single-chain Fv fragment; Se-2F3-scFv, selenium-containing 2F3-scFv.
- The Biochemical Society, London ©2001