PROLYL ISOMERASES CATALYZE ANTIBODY FOLDING IN-VITRO

Some slow-folding phases in the in vitro refolding of proteins origina te from the isomerization of prolyl-peptide bonds, which can be accele rated by a class of enzymes called prolyl isomerases (PPIs). We used t he in vitro folding of an antibody Fab fragment as a model system to s tudy the effect of PPI on a folding reaction that is only partially re versible. We show here that members of both subclasses of PPIs, cyclop hilin and FK 506 binding protein (FKBP), accelerate the refolding proc ess and increase the yield of correctly folded molecules. An accelerat ion of folding was not observed in the presence of the specific inhibi tor cyclosporin A, but still the yield of correctly folded molecules w as increased. Bovine serum albumin (BSA) increased the yield comparabl e to cyclophilin but, in contrast, did not influence the rate of react ivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate- limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several min utes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalys ts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds. Proline isomerization is obviously a late fo lding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction. (2) PPI and BSA are able to increase the yield of refolding of Fab by reducing the formation of aggregates or the adsorption to the surface of the reaction vessel in an unspecific manner. This behavior is clearly distinct from the mech anism of action observed with the chaperone GroEL.