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目的:探索Pfs25蛋白在毕赤酵母中高效分泌表达策略,提高Pfs25蛋白的表达量。方法:本研究主要通过4个方面深入研究Pfs25蛋白的高效表达策略。①构建Pfs25蛋白的诱导型表达重组株pPICZαA-Pfs25/GS115和组成型表达重组株pGAPZαA-Pfs25/GS115,并比较两酵母菌株的表达量差异。②从培养基、pH值、表达时间等方面入手,分别对诱导型和组成型分泌表达条件进行研究,寻找适合Pfs25蛋白表达的最佳条件。③利用pAO815表达载体构建Pfs25多拷贝重组酵母菌株pAO815-(α-Pfs25)n/GS115,通过提高pfs25基因拷贝数来提高表达量。④将毕赤酵母中蛋白二硫键异构酶(PDI)基因引入Pfs25重组酵母中,以提高Pfs25蛋白的分泌表达。结果:本研究成功构建了Pfs25诱导型和组成型表达重组酵母菌株,实现了Pfs25的诱导型和组成型表达,组成型表达量略高于诱导型。并通过对各种表达条件的比较研究,找到了适合Pfs25蛋白在毕赤酵母中高效分泌表达的条件。结论:通过对Pfs25蛋白高效表达策略的研究,显著提高了Pfs25蛋白在毕赤酵母中的分泌表达水平,使其表达量提高了约5倍。
Objective: To explore the strategy of high efficient secretion of Pfs25 protein in Pichia pastoris and increase the expression of Pfs25 protein. Methods: This study mainly through four aspects of in-depth study Pfs25 protein expression strategy. ① Construction of inducible Pfs25 protein expression recombinant pPICZαA-Pfs25 / GS115 and constitutively expressed recombinant pGAPZαA-Pfs25 / GS115, and to compare the differences in the expression levels of the two yeast strains. (2) To study the conditions of inducible and constitutive secretory expression from the aspect of culture medium, pH value and expression time, and find out the optimal conditions for Pfs25 protein expression. (3) The pAO255 multi-copy recombinant yeast strain pAO815- (α-Pfs25) n / GS115 was constructed by using the pAO815 expression vector, and the expression was enhanced by increasing the pfs25 gene copy number. ④ The Pichia pastoris protein disulfide isomerase (PDI) gene introduced Pfs25 recombinant yeast, in order to improve the secretion of Pfs25 protein expression. Results: Pfs25 inducible and constitutively expressed recombinant yeast strains were successfully constructed in this study. The induction and constitutive expression of Pfs25 were achieved. The constitutive expression of Pfs25 was slightly higher than that of inducible. Through the comparative study of various expression conditions, the conditions for the efficient secretion of Pfs25 protein in Pichia pastoris were found. Conclusion: The high expression of Pfs25 protein in Pichia pastoris increased the secretive expression of Pfs25 protein by about 5-fold.