School of Biological and Chemical Engineering, Zhejiang University of Science & Technology, Hangzhou 310023, China.
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Biomed Res Int. 2018 Sep 27;2018:3560894. doi: 10.1155/2018/3560894. eCollection 2018.
Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid (-propargyloxyphenylalanine, Ppa) (P-AQP SS9) using a mutated tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (P) of 7.46×10 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (P=1.31×10 m/s) and 12.1 times higher than that of the DOPC liposomes (P=6.15×10m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.
水通道蛋白（AQP）广泛应用于仿生膜的水再循环和海水淡化中。本研究从 SS9 中分离出一种新型水通道蛋白（AQP SS9），其具有较高的水透过率，有望应用于实际的水净化。为了提高嵌入仿生膜中水通道蛋白 SS9 的稳定性，通过突变的酪氨酸 tRNA 合成酶（TyrRS）和无细胞表达系统，将非天然氨基酸（-炔丙基苯丙氨酸，Ppa）（P-AQP SS9）掺入到 AQP SS9 中，得到改良的 AQP SS9。修饰后的 AQP SS9 可以与磷脂共价连接，从而显著提高仿生膜的稳定性。评估了 Mg 的浓度和与信号肽的融合表达以提高 P-AQP SS9 的表达水平，最终产量最高可达 49mg/L。然后将修饰后的 AQP SS9 重新组装到 DOPC 脂质体中，并通过停流分光光度计进行分析。得到的水透过系数（P）为 7.46×10 m/s，比野生型 AQP SS9 的蛋白脂质体（P=1.31×10 m/s）高 5.7 倍，比 DOPC 脂质体（P=6.15×10m/s）高 12.1 倍。本研究展示了一种具有更高稳定性的膜蛋白无细胞表达系统的开发，以及修饰后的水通道蛋白在水过滤方面的潜在应用。
