肠激酶在毕赤酵母中的分泌表达优化  

Optimization of enterokinase secretion in Pichia pastoris

作　　者：梁启星 石竟成 金学荣 堵国成[1,2] 康振 Qixing Liang;Jingcheng Shi;Xuerong Jin;Guocheng Du;Zhen Kang(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2020年第8期1689-1698,共10页Chinese Journal of Biotechnology

基　　金：江南大学自主科研计划重点项目(No.JUSRP51707A);国家轻工技术与工程一流学科自主课题(No.LITE2018-16)资助。

摘　　要：肠激酶(Enterokinase,EK)是一类特异性识别切割DDDDK序列的丝氨酸蛋白酶,作为一种工具酶广泛应用于生物医药领域。目前,EK在毕赤酵母Pichiapastoris中的表达水平较低,难以应用。本研究比较了6种不同的信号肽SP1、SP2、SP3、SP4、SP7和SP8对毕赤酵母分泌表达EK的影响。在摇瓶水平上,与α-factor信号肽相比,SP1信号肽显著提高了EK的分泌表达(从6.8 mg/L提高至14.3 mg/L),酶活从(2390±212)U/mL提高至(4995±378)U/mL。在此基础上,通过共表达毕赤酵母内源蛋白Kex2,EK酶活提高至(7219±489)U/mL。另外,N端融合WLR三个氨基酸进一步提高酶活至(15145±920)U/mL,比酶活为(1174600±53100)U/mg。EK在毕赤酵母中的高效分泌表达为未来应用奠定了基础。Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences.Therefore,enterokinase has been widely used as a tool enzyme in the field of biomedicine.Currently,the expression level of enterokinase in Pichia pastoris is low,which hinders related practical applications.In this study,the effects of six different signal peptides SP1,SP2,SP3,SP4,SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied.Compared withα-factor,SP1 significantly increased the secretory expression of enterokinase(from 6.8 mg/L to 14.3 mg/L),and the enterokinase activity increased from(2390±212)U/mL to(4995±378)U/mL in shaking flask cultures.On this basis,the enterokinase activity was further enhanced to(7219±489)U/mL by co-expressing the endogenous protein Kex2.Moreover,the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to(15145±920)U/mL with a high specific activity of(1174600±53100)U/mg.The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

关 键 词：毕赤酵母 肠激酶 信号肽 共表达 N端改造 

分 类 号：Q936[生物学—微生物学]