雅致放射毛霉羧肽酶Y在毕赤酵母中的高效分泌表达

High level secretory expression of Carboxypeptidase Y from Actinomucor elegans in Pichia pastoris

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.