Fogeron Marie-Laure, Paul David, Jirasko Vlastimil, Montserret Roland, Lacabanne Denis, Molle Jennifer, Badillo Aurélie, Boukadida Célia, Georgeault Sonia, Roingeard Philippe, Martin Annette, Bartenschlager Ralf, Penin François, Böckmann Anja
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France.
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
Protein Expr Purif. 2015 Dec;116:1-6. doi: 10.1016/j.pep.2015.08.027. Epub 2015 Aug 29.
Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.
丙型肝炎病毒（HCV）的非结构蛋白2（NS2）是一种整合膜蛋白，含有半胱氨酸蛋白酶，在感染性子代病毒粒子的组装中起核心组织作用。虽然蛋白酶结构域的晶体结构已被解析，但NS2全长形式在生化和结构上仍未得到表征，因为无法从基于细胞的系统中大量制备重组NS2。我们在此表明，在NS2-NS3pro前体蛋白背景下的功能性NS2（确保NS2-NS3裂解）可以通过使用麦胚无细胞表达系统有效表达。在同一系统中，我们随后成功生产并纯化了毫克量的呈预期二级结构含量的去污剂可溶解形式的全长NS2。此外，对重组蛋白脂质体的免疫电子显微镜分析表明NS2与模型膜相关联。
