Improvement Of Secretory Expression Of Fusion Protein HSA/IFNβ In Pichia Pastoris

The fusion protein composed of human serum albumin (HSA) and different drug peptideor protein had successfully been expressed in Pichia pastoris in our laboratory. However, pre-liminary study established numerous differences in expression level and the degradation ofHSA fusion proteins expressed in P. pastoris, although the same drug peptide conjugated tothe various terminals of HSA, N-terminal or C-terminal. In this study, we developed a methodto improve the expression of human serum albumin and interferon β fusion protein(HSA/IFNβ) in P. pastoris by genetic engineering technology.In this study, We adjusted the secretory passway of the fusion protein in P. pastorisGS115/pPIC9K-HSA-IFNβ by co-expressing the factors which effect secretory passway. Here,we analysed five factors: molecular chaperone, BiP; disulfide isomerase, PDI; oxidoreductas-es of the protein disulfide isomerase, Ero1; and the vesicle trafficking protein, Sec1and Sly1.Results indicated that factors co-expression were critical to obtain HSA/IFNβ50-90%higherthan control group and had no effect on the growth of P. pastoris. However, BiP co-expressionhad no improvement on production yields.Meanwhile, we studied the effect of inserting different linkers between HSA and IFNβ inP. pastoris. We designed four linkers with different properties according to the length andstructure of the linkers molecular: RL, HL, SL and LL. The results showed that: the short andrigid linker, RL, could improve the expression level of HSA/IFNβ significantly (about2.2-folds compared with the control); and the long linker, LL, almost had no effect on theexpression of fusion protein; but when using the other two linkers, HL and SL, the expressionof fusion protein decreased about30%. Linkers could not help to reduce degradation, and HLand SL even could make HSA/IFNβ easier to degrade in P. pastoris.Furthermore, we transformed the proteasome system of P. pastoris and expressed the fu-sion protein HSA/IFNβ in yps1Δpep4Δ double disruptant strain. Protease gene YPS1&PEP4were successfully knocked out. The degradation was significantly reduced68%inyps1Δpep4Δ protease double disruptant strain compared with the wild strain. And there wasno influence on its normal growth.