PURIFICATION OF A RECOMBINANT HUMAN RESPIRATORY SYNCYTIAL VIRUS CHIMERIC GLYCOPROTEIN USING REVERSED-PHASE CHROMATOGRAPHY AND PROTEIN REFOLDING IN GUANIDINE-HYDROCHLORIDE

FG glycoprotein is a recombinant chimeric protein consisting of the ex tracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be eff ective as a RSV vaccine. Recombinant FG glycoprotein was expressed usi ng the baculovirus/insect cell system. FG glycoprotein was isolated fr om cell culture supernatants using S Sepharose ion-exchange chromatogr aphy, Cu2+-immobilized metal affinity chromatography, preparative reve rsed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergen t. The purified FG glycoprotein was concentrated on a S Sepharose colu mn and exchanged into an appropriate buffer for vaccine formulation. F ive batches of FG glycoprotein with protein purity of 92-99% were prod uced using this purification process. FG glycoprotein produced using r eversed-phase chromatography and protein refolding was compared with n ondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G gly coproteins. The results of these studies indicated that refolded FG gl ycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein. (C) 1994 Academic Press, Inc.