Cloning and expression of a single-chain catalytic antibody that acts as aglutathione peroxidase mimic with high catalytic efficiency

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxy gen species. In previous papers we have developed a new strategy for genera ting abzymes: the monoclonal antibody with a substrate-binding site is firs t prepared, then a catalytic group is incorporated into the monoclonal anti body's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, L iu, Yang and Shen (1994) Biochem. Biophys. Res, Commun. 198, 1240-1247; Din g, 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 ca talytic efficiency, exceeding that of rabbit liver GPX, after chemical muta tion. To produce pharmaceutical proteins and to study the reason why it exh ibits high catalytic efficiency, we sequenced, cloned and expressed the var iable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in d ifferent bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%,15-20 % and 25-3 0 % of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co2+-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in v itro by gel filtration. The binding constants of the active 2F3-scFv for GS H and GSSG were 2.46 x 10(5) M-1 and 1.03 x 10(-5) M-1 respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. T he 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 3394 units/mu mol, which approaches the activity of rab bit liver GPX.