肠激酶在毕赤酵母中的分泌表达优化

Optimization of enterokinase secretion in Pichia pastoris

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Pichia pastoris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 (2 390±212) U/mL提高至 (4 995±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 (7 219±489) U/mL。另外，N端融合WLR三个氨基酸进一步提高酶活至 (15 145±920) U/mL，比酶活为 (1 174 600±53 100) 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。

关键词:毕赤酵母，肠激酶，信号肽，共表达，N端改造

Abstract:

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 with α-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 (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±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 (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

Key words:Pichia pastoris, enterokinase, signal peptide, co-expression, N-terminal modification