Xu Zhinan, Lian Jiazhang, Cai Jin
Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
Protein Pept Lett. 2010 Feb;17(2):181-5. doi: 10.2174/092986610790225996.
Aquaporin Z (AqpZ) is a typical orthodox aquaporin with 6 transmembrane domains and five connecting loops. In order to express this complex membrane protein efficiently, E. coli cell-free expression system was employed as an alternative to produce aquaporin Z. Using different fusion vectors containing AqpZ gene, the expression level of fusion proteins in cell-free system varied from 7.97 to 578.35 microg/ml, while 7.34 to 340.81 microg/ml for target protein (AqpZ). The free energy of mRNA secondary structure at translation initiation region (TIR) was predicted and demonstrated a positive relationship with the expression level of AqpZ in cell-free system. This is the first report of expressing water channel protein in E. coli cell-free system, which has become a highly promising tool for fast and efficient production of integral membrane proteins.
水通道蛋白Z（AqpZ）是一种典型的具有6个跨膜结构域和5个连接环的正统水通道蛋白。为了高效表达这种复杂的膜蛋白，采用大肠杆菌无细胞表达系统作为替代方法来生产水通道蛋白Z。使用含有AqpZ基因的不同融合载体，无细胞系统中融合蛋白的表达水平在7.97至578.35微克/毫升之间变化，而目标蛋白（AqpZ）的表达水平为7.34至340.81微克/毫升。预测了翻译起始区域（TIR）处mRNA二级结构的自由能，并证明其与无细胞系统中AqpZ的表达水平呈正相关。这是在大肠杆菌无细胞系统中表达水通道蛋白的首次报道，该系统已成为快速高效生产整合膜蛋白的极具前景的工具。
