分别构建以2种启动子表达雅致放射毛霉羧肽酶Y的重组毕赤酵母菌株GS115,通过提高抗生素浓度筛选到具有不同拷贝数羧肽酶Y基因的重组菌株,通过对蛋白表达量的比较筛选出高效分泌表达菌株。在筛选得到的重组菌株中共表达伴侣蛋白binding protein(BIP)、protein disulfide isomerase(PDI)、endoplasmic oxidoreductin 1(ERO1),分别将羧肽酶Y酶原的分泌表达量提高到1.87倍、1.72倍和1.26倍。体外羧肽酶Y酶原可以由胰蛋白酶激活。该研究实现了雅致放射毛霉羧肽酶Y在毕赤酵母中的高效分泌表达,为其工业化应用提供了技术支持。
Herein recombinant Pichia pastoris strain GS115 was constructed to express Actinomucor elegans carboxypeptidase Y under controlling of two promoters and a series of recombinant strains with different copy numbers were screened by continuously increasing the antibiotic concentration. The co-expression of the chaperone protein binding protein(BIP), protein disulfide isomerase(PDI) and endoplasmic oxidoreductin 1(ERO1) were respectively used as co-expressed chaperone protein in the screened recombinant strains and increased the secretory expression of carboxypeptidase Y zymogen by 1.87, 1.72, and 1.26 times, respectively. Carboxypeptidase Y zymogen can be activated by trypsin in vitro. In this study, Actinomucor elegans carboxypeptidase Y was highly expressed in Pichia pastoris and technical support was provided for its industrial application.
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