膜结合水通道蛋白（水通道蛋白Z）在大肠杆菌中的高效表达。

Efficient expression of membrane-bound water channel protein (Aquaporin Z) in Escherichia coli.

作者信息

Lian Jiazhang, Fang Xiangming, Cai Jin, Chen Qixing, Zheng Qiang, Kai Lei, Xu Zhinan

机构信息

Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.

出版信息

Protein Pept Lett. 2008;15(7):687-91. doi: 10.2174/092986608785133717.

DOI:10.2174/092986608785133717

PMID:18782063

Abstract

In order to explore the possibility of preparing a high-efficiency aquaporin-based biofilter, an efficient approach for expression of membrane-bound Aquaporin Z (AqpZ) in E. coli was proposed. The AqpZ gene was amplified by means of PCR, and two expression vectors (pET28-AqpZ and pET32-AqpZ) were constructed. The channel protein of interest was synthesized in E. coli BL21(DE3)/pET32-AqpZ as an insoluble fusion protein linked with trxA. However, with BL21(DE3)/pET28-AqpZ, significant amount of AqpZ fused only with 6-His (6-His-AqpZ) could be expressed, correctly folded and targeted into the membrane. Under the optimized culture conditions, the highest expression level (9.05 mg/l) of membrane-bound 6-His-AqpZ was achieved with BL21(DE3)/pET28-AqpZ, and an additional amount (2.35 mg/l) was expressed concomitantly as the inclusion body form. This expression result was 3.5 times higher than that in the previous studies.

摘要

为了探索制备基于水通道蛋白的高效生物滤器的可能性，提出了一种在大肠杆菌中高效表达膜结合水通道蛋白Z（AqpZ）的方法。通过PCR扩增AqpZ基因，并构建了两个表达载体（pET28-AqpZ和pET32-AqpZ）。目的通道蛋白在大肠杆菌BL21(DE3)/pET32-AqpZ中作为与硫氧还蛋白（trxA）相连的不溶性融合蛋白合成。然而，在BL21(DE3)/pET28-AqpZ中，大量仅与6-组氨酸（6-His-AqpZ）融合的AqpZ能够表达、正确折叠并靶向到膜中。在优化的培养条件下，BL21(DE3)/pET28-AqpZ实现了膜结合6-His-AqpZ的最高表达水平（9.05 mg/l），同时还以包涵体形式表达了额外的量（2.35 mg/l）。该表达结果比先前研究高3.5倍。