重组人血清白蛋白-干扰素α2b融合蛋白在PichiaPink系统中的表达

摘要

Objective: The aim of this experiment was to compare the differences between the PichiaPink strain and Pichia pastoris GS115 strain, and further explore the potential advantages of the PichiaPink system. Methods: The recombinant expression vectors contained the fusion gene coding human serum albumin and interferon α2b fusion protein (HAS-IFN-α2b) were constructed, and were transformed them into the P.pastoris GS115 and the PichiaPink strain respectively. The fusion protein of HAS-IFN-α2b were expressed by methanol induction, and the expression level was evaluated. The SDS-PAGE analysis was used to detect the degradation of HAS-IFN-α2b in the P.pastoris GS115 and the PichiaPink strain. Results: Essentially all transformants in the PichiaPink system showed to express the objective protein of HAS-IFN-a2b, but only 60% transformants in the GS115 strain displayed to express the target protein. In the same kind strains of Pink, the expression levels of HAS-IFN-α2b were respectively compared, which discovered the strain that have integrated with the pPink-HC vector is higher expression than the strain that have integrated with the pPink-LC vector. The PichiaPink strains which had knockouted three proteases genes expressed the fusion protein of HAS-IFN-α2b to exhibit a little degradation in YPD and BMMY medium, but HAS-IFN-α2b degradation phenomenon showed very serious in BSM medium. Conclusion: The results have indicated to have an important value for high-level expression and large-scale production of secreted recombinant proteins in PichiaPink system.%目的:比较PichiaPink表达系统和巴斯德毕赤酵母GS115在表达外源蛋白方面的差异,对PichiaPink表达系统的潜在优点进行评价.方法:以重组人血清白蛋白-干扰素α2b融合蛋白(HSA-IFN-α2b)为报告蛋白,构建相关载体,分别转化PichiaPink系统菌株和巴斯德毕赤酵母GS115菌株,诱导HSA-IFN-α2b表达并测定表达水平；通过SDS-PAGE检测HSA-IFN-α2b在PichiaPink系统中的降解程度.结果:PichiaPink系统几乎所有的转化子都可以表达HSA-IFN-α2b,而GS115菌株只有60％的转化子能表达HSA-IFN-α2b;同一种Pink菌株中,整合有高拷贝数表达载体pPink-HC的菌株表达量高于整合有低拷贝数表达载体pPink-LC的菌株；Pink蛋白酶缺陷菌株在复杂培养基(YPD,BMMY)中HSA-IFN-α2b基本没有降解,而在合成培养基(BSM)中降解现象明显.结论:PichiaPink表达系统产生的转化子较GS115菌株产生的转化子易于筛选；PichiaPink系统蛋白酶缺陷菌株可明显减少外源蛋白降解.这些结果为利用PichiaPink表达系统高水平和大规模制备外源蛋白提供了实验依据.