Cloning Of Pichia Pastoris YPS2 And Construction Of PpYPS2 Null Mutant

As the representative of the second generation of yeast expression system, Pichia Pastoris, a methylotrophic yeast, has become a highly successful and sub-consummate eukaryotic expression system for its high expression, stability and secretion. It possesses many advantages that other expression systems don't have and has been applied to not only industrial manufactures but also foundational researches. But this expression system also have some shortcomings, such as some heterogenous proteins are degraded by endogenous protease in yeast during its secretion expression, that limit its far-flung applications.YPS2 (yeast aspartic protease 2), one of yeast aspartic proteases, cleaves specifically mono- or di-basic sites at C-terminal and plays a role in the processing of the propeptides. Our previous studies indicated the cleavage of the foreign protein mostly occur at dibasic site, so it is supposed that this degradation was possibly related to yapsin proteinase family. Furthermore, our previous study had also showed the degradation of HSA-AX15(R13K) when expressed in YPS1? strain was obviously improved. But no improvement in the degradation of HSA-IFN-α2b was found when it was expressed in this protease deficient strain. Therefore, in this study, we plan to construct the YPS2 deficient strain to solve the degradation of foreign protein when expressed in Pichia pastoris.In this study, YPS2 gene was isolated from Pichia pastoris via CODEHOP PCR, IPCR and TAIL-PCR methods, and then the YPS2 null mutant was constructed. Evalution was carried out for this protease deficient strain using reporter proteins, such as HSA-IFN-α2b, HSA-AX15(R13K) and HSA.The results showed that the degradation of HSA-IFN-α2b when expressed in the YPS2? strain wasn't improved, consistent with its expression in wild-type strain, while the degradation of HSA-AX15(R13K) was slightly improved. After the insoluble proteins from YPS1? and wild GS115 strains were incubated seperatedly with HSA, there was no difference in the degradation bands of HSA at a pH of 6.0, but a significant difference at a pH of 4.0 with complete degradation of HSA by the insoluble protein of wild GS115 strain and partial degradation by that of YPS1? strain. This result indicates that YPS2 is almost inactive at pH 6.0, but significantly active at pH 4.0.