Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system

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• 作者：Xu Zhang ; Jiazhang Lian ; Lei Kai ; Lei Huang ; Peilin Cen ; Zhinan Xu
• 关键词：Aquaporin Z ; Cell-free protein synthesis ; Leader peptide ; Leader peptidase ; Membrane protein
• 刊名：Enzyme and Microbial Technology
• 出版年：5 February, 2014
• 年：2014
• 卷：55
• 期：Complete
• 页码：26-30
• 全文大小：794 K

文摘

Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 卤 0.1) 脳 10^鈭?4 cm³ s^鈭? monomer^鈭?. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.