Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors

A cellular assay system for measuring the activity of cytoplasmically expre ssed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domai n was established in the budding yeast Saccharomyces cerevisiae. The inhibi tory potential of different constitutively expressed anti-GCN4 scFv intrabo dies was monitored by measuring the activity of beta-galactosidase expresse d from a GCN4-dependent reporter gene. The in vivo performance of these scF v intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant induced equilibrium un folding. A framework-engineered stabilized version showed significantly imp roved activity, while a destabilized point mutant of the anti-GCN4 wild-typ e showed decreased effects in vivo. These results indicate that stability e ngineering can result in improved performance of scFv fragments as intrabod ies. Increasing the thermodynamic stability appears to be an essential fact or for improving the yield of functional scFv in the reducing environment o f the cytoplasm, where the conserved intradomain disulfides of antibody fra gments cannot form.