Isotopically labeled expression in E-coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein
Isotopically labeled expression in E-coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein
复制标题
DOI：
10.1016/j.pep.2008.06.009
复制
发表时间：
2008-10-01
影响因子：
1.6
通讯作者：
Weliky, David P.
中科院分区：
文献类型：
--
作者：
Curtis-Fisk, Jaime;Spencer, Ryan M.;Weliky, David P.
本文报道了大肠杆菌摇瓶培养生产同位素标记的23 kDa病毒膜蛋白的方法，其纯度为20 mg/L。这一产率足以用于核磁共振结构研究，并且蛋白质的制备方法简单、直接、快速，可能适用于在大肠杆菌中表达的其他重组膜蛋白。目标FHA2蛋白是流感病毒血凝素蛋白的全胞外域结构，在感染过程中催化病毒和细胞内体膜之间的融合。FHA2的高产是通过以下步骤实现的：(1)在富含培养基中初始生长至类似于A(600)，然后切换到最低限度的培养基并诱导蛋白质表达；(2)通过洗涤剂可溶裂解产物的纯化和包涵体的增溶、纯化和复性获得蛋白质。通过对pH值、氧气量、碳源和浓度的优化，获得了较高的细胞密度，并利用圆二色谱优化了复性方案。对于通过包涵体纯化和复性得到的膜结合FHA2的单一残基，通过固体核磁共振光谱测量的(CO)-C-13化学位移证实了其天然构象。(C)2008 Elsevier Inc.保留所有权利。
This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A(600)similar to 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the (CO)-C-13 chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy. (c) 2008 Elsevier Inc. All rights reserved.