Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan, Taiwan, Republic of China.
International Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Curr Protoc Mol Biol. 2020 Sep;132(1):e126. doi: 10.1002/cpmb.126.
Transmembrane proteins are responsible for many critical cellular functions and represent one of the largest families of drug targets. However, these proteins, especially multipass transmembrane proteins, are difficult to study because they must be embedded in a lipid bilayer to maintain their native conformations. The development of the virion display (VirD) technology enables transmembrane proteins to be integrated into the viral envelope of herpes simplex virus 1 (HSV-1). Combining high-throughput cloning, expression, and purification techniques, VirD technology has been applied to the largest set of human transmembrane proteins, namely G-protein-coupled receptors, and has allowed the identification of interactions that are both specific and functional. This article describes the procedures to integrate an open reading frame for any transmembrane protein into the HSV-1 genome and produce recombinant HSV-1 virus to ultimately generate pure VirD virions for biological and pharmaceutical studies. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gateway cloning of transmembrane proteins Support Protocol 1: Ethanol precipitation of bacterial artificial chromosomal DNA Support Protocol 2: Preparation of competent cells Basic Protocol 2: Production of recombinant HSV-1 virions.
跨膜蛋白负责许多关键的细胞功能，是最大的药物靶点家族之一。然而，这些蛋白质，特别是多穿膜跨膜蛋白，由于必须嵌入脂质双层才能保持其天然构象，因此很难研究。病毒展示（VirD）技术的发展使跨膜蛋白能够整合到单纯疱疹病毒 1（HSV-1）的病毒包膜中。结合高通量克隆、表达和纯化技术，VirD 技术已应用于最大的一组人类跨膜蛋白，即 G 蛋白偶联受体，并能够鉴定出特异性和功能性的相互作用。本文描述了将任何跨膜蛋白的开放阅读框整合到 HSV-1 基因组中并产生重组 HSV-1 病毒的步骤，最终生成用于生物学和药物研究的纯 VirD 病毒颗粒。© 2020 威利父子公司。基本方案 1：跨膜蛋白的 Gateway 克隆 支持方案 1：细菌人工染色体 DNA 的乙醇沉淀 支持方案 2：感受态细胞的制备 基本方案 2：重组 HSV-1 病毒粒子的生产。
