Ooi Amanda, Wong Aloysius, Esau Luke, Lemtiri-Chlieh Fouad, Gehring Chris
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology Thuwal, Saudi Arabia.
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and TechnologyThuwal, Saudi Arabia; Institute of Integrative Biology of the Cell, Centre National de la Recherche Scientifique, Le Commissariat à l'Energie Atomique et aux Energies Alternatives, Paris-Sud UniversityGif-Sur-Yvette, France.
Front Physiol. 2016 Jul 19;7:300. doi: 10.3389/fphys.2016.00300. eCollection 2016.
The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K(+) channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.
人胚肾293（HEK - 293）细胞常用于膜蛋白的异源表达宿主，这主要是因为它们具有高转染效率，能准确翻译和加工蛋白质。此外，它们的细胞大小、形态和分裂速率，以及天然通道的低表达，这些特性对于电流 - 电压测量特别有吸引力。然而，对于诸如受体和离子通道等复杂膜蛋白的异源表达，用于生物学特性研究，特别是用于单细胞应用（如电生理学）仍然是一个挑战。功能性蛋白质的表达很大程度上取决于精心的逐步优化，这包括设计带有合适识别标签的表达载体，以及选择适合该应用的转染方法和检测参数。在这里，我们以植物钾通道拟南芥保卫细胞外向整流钾通道AtGORK（At5G37500）在HEK - 293细胞中的异源表达为例，评估常用的转染试剂和荧光检测方法，并提供一种详细的方法，用于优化膜蛋白的瞬时转染和表达，以用于一般的体内研究，特别是单细胞应用。这种优化方案将有助于复杂膜蛋白的生理学和细胞特性研究。
