The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou 22116, China.
Department of Biology, Applied Plant Sciences, Technische Universität Darmstadt, Schnittspahn Strasse 10, D-64287 Darmstadt, Germany.
Cells. 2019 Oct 27;8(11):1325. doi: 10.3390/cells8111325.
Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from and using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and PIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.
水通道蛋白是重要且研究充分的膜通道蛋白。然而，作为膜蛋白，用于功能分析的样品制备既繁琐又耗时。在本文中，我们报告了一种新的方法，用于使用 CFPS 系统共翻译插入来自 和 的两种水通道蛋白。这是在存在脂质体的情况下完成的，采用了改良的程序来形成均质的蛋白脂体，适用于使用停流分光光度法进行水通透性的功能分析。两种模型水通道蛋白 AqpZ 和 PIP2;1 成功地以其活性形式掺入到脂质体中。绿色荧光蛋白的突变体被融合到 AqpZ 的 C 末端，以监测其在脂质环境中的插入和状态。这种新的快速方法为在原核和真核生物中对水通道蛋白进行功能分析提供了一种快速而直接的方法。
