Abstract:

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus(HEV). HEV is a nonenveloped virus that has been classified in the family of Caliciviridae. The virus appears to be a polyadenylated, positive-stranded RNA virus with three major open reading frames(ORFs). The capsid protein of HEV is encoded by the open reading frame 2(ORF2). We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids(aa) 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P. pastoris. Under optimized conditions (culture medium pH, 6.0―6.5; methanol concentration added daily, 3.0%; inoculum density, OD₆₀₀=60; induction time point, 72―96 h), the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.

Key words: Hepatitis E virus; Capsid protein; Pichia pastoris; Protein purification