PURIFICATION AND RENATURATION OF JAPANESE ENCEPHALITIS-VIRUS NONSTRUCTURAL GLYCOPROTEIN NS1 OVERPRODUCED BY INSECT CELLS

The nonstructural protein NS1 of Japanese encephalitis virus is a majo r immunogen produced during flavivirus infection. However, the functio n of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle , the protein was produced in Spodoptera frugiperda insect cells (Sf9) , using a recombinant baculovirus, and purified. As described previous ly by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-83 6), a small fraction of the synthesized recombinant protein could matu re into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a pur ified form using a two-step procedure. Isolated inclusion bodies, in w hich NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promotin g reoxidation-renaturation of the purified protein were then investiga ted at a concentration of 100 mu g/ml at pH 8. The presence of 8 M ure a during reoxidation of NS1 with oxidized glutathione was essential pr ior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in. vitro. Three major species, all monomeric, we re resolved by nonreducing SDS-PAGE. The form showing the lowest appar ent molecular weight comigrated with native unreduced NS1 and was reco gnized by a monoclonal antibody directed against a conformational epit ope strictly dependent on the native structure of the protein. Thus, t his form, representing over 30% of the renatured products, may have re ached the native conformation of the protein. This simple renaturation -reoxidation procedure may be applicable to other disulfide bond-conta ining proteins to be recovered in the native state from inclusion bodi es. (C) 1995 Academic Press, Inc.