Abstract：
Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.
摘要：
转运蛋白的功能表征受到当前转运蛋白测定的高成本和技术挑战的阻碍。因此，在这项工作中，我们开发了一种新的表征工作流程，该工作流程结合了无细胞蛋白质合成（CFPS）和固体支持的基于膜的电生理学（SSME）。为此，膜蛋白合成是在纳米圆盘存在下在连续交换无细胞系统（CECF）中完成的。将在纳米盘中表达的所得转运体掺入蛋白脂质体中，并在不同底物存在下使用表面电事件读取器进行测定。作为概念的证明，我们验证了这个工作流程来表达和表征五种不同的转运蛋白：药物/H+偶联的反转运蛋白EmrE和SugE，乳糖渗透酶LacY，来自大肠杆菌的Na+/H+反转运蛋白NhaA，和来自酿酒酵母的线粒体载体AAC2。对于所有运输机动力学参数，比如KM，IMAX,和pH依赖性，进行了评估。这种强大而快速的工作流程(例如，可以在仅五个工作日内执行）提供了对转运蛋白活性的方便直接功能评估，并且能够促进转运蛋白在医学和生物技术研究中的应用。
