Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

摘要

Glutathione peroxidase(GPX) plays an important role in scavenging reactive oxygen species. A series of cataly-{tic} antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, %S%-2,4-dinitriphenyl {%t%-butyl} ester(GSH-s-DNP-Bu) and %S%-2,4-dinitriphenyl %t%-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.