Efficient Expression Of The Escherichia Coli Water Channel Protein (Aquaporin Z) In Vivo And In Vitro

Aquaporin Z (AqpZ), the water channel protein from Escherichia coli, is an orthodox aquaporin with high water selectivity and high permeability, making AqpZ-based water filter a promising tool for water reuse and seawater desalination. However, the development of this novel bio-filter is hampered by the availability of sufficient amount of water channel proteins since the overexpression of integral membrane proteins may exhibit toxicity towards the host due to the highly hydrophobic nature.The aim of this work is to synthesize functional AqpZ at high levels either by fusion expression strategy or cell-free expression system, which lays a solid foundation to construct the novel aquaporin-based water filter, and also promises a universal platform for efficient production of membrane proteins.Firstly, fusion expression was chosen to improve the expression level of AqpZ. By fusion with several hydrophilic partners, the yield of AqpZ was significantly improved, and MBP was determined to be the most efficient fusion partner to increase the expression level. After systematically investigating the effects of different induction conditions, such as temperature, induction-timing, IPTG concentration and post-induction duration, high productivity of MBP-AqpZ (200 mg/l) was achieved.Because the overexpression of membrane proteins would lead to cell toxicity, no growth-required cell-free protein synthesis (CFPS) system was employed as an alternative to synthesize AqpZ. Several in vitro expression vectors with different N-terminal sequence were constructed, and pIVEX2.4c-AqpZ was determined to be the most efficient vector in cell-free system. However, due to the lack of hydrophobic environment essential for membrane protein folding, AqpZ was aggregated as precipitates in a standard CFPS. In order to produce soluble AqpZ, detergent resolubilization was investigated first. However, all the tested detergents in this work failed to resolubilize the AqpZ precipitates efficiently. Therefore, detergents were supplemented directly instead, taking advantage of the open nature of CFPS. By systematically screening and optimizing of detergent structure and concentration, the highest soluble expression (530 mg/1) was achieved by Brij78 at a final concentration of 112xCMC. In addition, DOPC liposome supplementation was also investigated, and 470 mg/1 membrane-associated AqpZ was achieved at the highest DOPC concentration tested (5 mg/ml).Furthermore, the formation of mRNA secondary structure was found to inhibit translation initiation, which limited the expression level of native AqpZ in cell-free system. Therefore, research work on two-cistronic plasmid strategy and leader peptide strategy were undertaken. Although the constructed two-cistronic plasmids could improve the expression level of AqpZ in vivo, these plasmids didn't work well in cell-free system. On the contrary, by fusion with many naturally occurring leader peptides, the expression level of AqpZ was enhanced by as much as 5 times through avoiding the formation of secondary structure. In addition, the leader peptide could be cleaved in situ in cell-free system supplemented with detergent or liposome, through the activation of leader peptidase. These results indicated that leader peptide sequence could be used as a downstream box to stimulate recombinant protein synthesis in cell-free system, promising a universal platform for efficient production of membrane proteins, regardless of the nature of target DNA sequence.