Expression and purification of dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been used as a host to expre ss the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a ch imera with hepatitis B surface antigen (HBsAg): a protein known to self ass emble into virus-like particles (VLPs) and to be efficiently expressed in P . pastoris. The Den2E gene used in this study is a truncated version encodi ng the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The f irst one contains the HBsAg gene as the 5' partner and the Den2E gene as th e 3' partner (HBsAg-Den2E). In the second one, the relative positions of th e two partners of the gene fusion were reversed to create the hybrid Den2E- HBsAg gene. These fusion genes were integrated into the genome of P. pastor is under the control of the methanol-inducible alcohol oxidase (AOX1) promo ter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher level s in P. pastoris based on Northern analysis. The hybrid protein (similar to 68 kDa) expressed by this clone was purified to near homogeneity using a c ombination of acid precipitation, hydrophobic interaction, and immunoaffini ty chromatographic steps. Final purification achieved was similar to 1400-f old with a yield of similar to 26%. The chimeric protein was found to posse ss the ability to assemble into high molecular weight aggregates (akin to H BsAg particles). The recombinant fusion protein eluted close to the void vo lume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a posit ion very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeri c protein. (C) 2001 Academic Press.