Cloning and expression of the external-glycoprotein gene mutant from HIV-2in the methylotrophic yeast Pichia pastoris and identification of the glycoprotein

To achieve high-level expression of HIV-2(ROD) external glycoprotein gp105 in Pichia pastoris, the gp105 gene mutant tPI, with the 5 ' non-functional region of the gp105 gene removed, was obtained by PCR amplification and was cloned into secreted expression vector pHILSI. The His(+)Mut(s) recombinan t P. pastoris strain was screened by PCR and induced by methanol. SDS/PAGE and Western-blot analyses showed that mutation of the low-usage codon AGG i nto synonymous codon CGA and the introduction of the optimal codon TTC made P. pastoris overexpress tPI, an 85 kDa heterologous glycoprotein that was secreted into the medium and recognized specifically by HIV-2 polyclonal an tibody. The recombinant strain GS115/tPI had excellent genetic stability in terms of the properties of growth and expression of gp105, and seven out o f 58 recombinant stains with a yield of 29% were selected to be used for fu rther purification of gp105.