Optimization of enterokinase secretion in Pichia pastoris.

Qixing Liang; Jingcheng Shi; Xuerong Jin; Guocheng DU; Zhen Kang

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Optimization of enterokinase secretion in Pichia pastoris.

Author: Qixing LIANG ¹ ; Jingcheng SHI ¹ ; Xuerong JIN ¹ ; Guocheng DU ¹ ; Zhen KANG ¹
Author Information

1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: N-terminal modification; Pichia pastoris; co-expression; enterokinase; signal peptide
From: Chinese Journal of Biotechnology 2020;36(8):1689-1698
CountryChina
Language:Chinese
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.