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DsbA-DsbAmut fusion chaperon improved soluble expression of human trypsinogen-1 in Escherichia coli

Frontiers of Chemical Science and Engineering 2015年第4期9卷 511-521页

作者： Ye Liu Wenyong Zhang Xubin Yang Guangbo Kang Damei Wang He Huang Key Laboratory of System Bioengineering Department of Biochemical Engineering School of Chemical Engineering and Technology Tianjin University Tianjin 300072 China Collaborative Innovation Center of Chemical Science and Engineering Tianjin 300072 China College of Food Engineering and Biotechnology Tianjin University of Science and Technology Tianjin 300072 China Gan & Lee Pharmaceuticals Beijing 101100 China Taiyuan Institute of Technology Taiyuan 030000 China

A co-expressing system ofDsbA-DsbAmut was suggested for the first time to enhance the soluble expression of human trypsin-1. As a control, leaderless DsbA chaperone was also co-expressed with human trypsin-1. Vectors pET39b-trypsin and pET28a-DsbA- DsbAn^ut-trypsin with the above two DsbA fusion tag were constructed. The strain with vector pET39b-trypsin expressed fusion protein DsbA-trypsin in form of inclusion bodies. While in E. coli BL21 （DE3） strain with vector pET28a-DsbA-DsbAmUt-trypsin, the soluble expression of trypsin fusion protein was achieved. Under the optimized expression conditions, the soluble fraction accounted for about 49.43% of total DsbA-DsbAmUt-trypsin proteins in crude supernatant. The purification yield was 4.15% by nickel chelating chromatography and 3.3 mg activated trypsin with a purity of 88.68% was obtained from 1 L LB broth. To detect the possible functions of DsbA series chaperons in trypsin fusion protein, we analyzed the primary three-dimensional structure of fusion proteins, mainly focusing on the compatibleness between trypsin and fusion chaperons. The results suggested that （1） besides the primary function in periplasm, leaderless DsbA or DsbAmut may also act as a signal sequences-like leader targeted to periplasm that partly relieved the pressure from fusion protein overexpression and inclusion body formation, and （2） as there was significant soluble expression of DsbA-DsbAmUt-trypsin compared with DsbA-trypsin, DsbArout may function as charge or hydrophobic balance in recombinant protein DsbA-DsbAmut- trypsin.

关键词： DsbA DsbA-DsbAmut soluble expression,trypsin chaperon

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