Purification and renaturation of Japanese encephalitis virus nonstructural glycoprotein NS1 overproduced by insect cells.
The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function 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 previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which 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 promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 micrograms/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Flamand
M
M
Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur, Paris, France.
Chevalier
M
M
Henchal
E
E
Girard
M
M
Deubel
V
V
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D004664:Encephalitis Virus, Japanese / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007120:Immunochemistry; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D004220:Disulfides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins