Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris

Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.