Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed aga inst L-asparaginase (ASNase) of Escherichia coli were selected from a synth etic phage-display scFv library. The scFv46 fragment could enhance the resi stance of ASNase to trypsin proteolysis, with 70 % of the initial ASNase ac tivity present after the ASNase-scFv46 complex had been treated with trypsi n for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression v ector pET-21a and expressed at high levels (about 45 % of total cell protei n) in E. coil BL21 (DE3) as inclusion bodies. The refolded and purified scF v46 fragment was proved to protect ASNase, and the protective effect was fu rther confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residua l activity of the ASNase-scFv46 complex could reach about 78 % after treatm ent with trypsin for 30 min at 37 degrees C, The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.