PDF(Pubmed)
Abstract：
Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7TIR and MBP-tagged TLR3/5/7/8TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.
摘要：
Toll样受体(TLR)是先天免疫系统中的关键参与者。尽管在TLR结构生物学方面做出了巨大的努力，今天我们只知道四个人TLR细胞内TIR结构域的空间结构。它们都属于受体的五个亚家族之一。主要瓶颈之一是高水平生产可溶性形式的正确折叠的蛋白质。在这里,我们使用合理的方法来寻找在大肠杆菌细胞中以可溶形式产生所有十个人TLR家族成员的TIR结构域的最佳参数。我们表明可以产生数十毫克的可溶性His标记的TLR2/3/6/7TIR和MBP标记的TLR3/5/7/8TIR。我们还开发了纯化方案，并通过CD和NMR光谱证明，纯化的TLR2/3/7TIR证明了TIR域固有的结构组织。这说明了所产生的蛋白质的正确折叠及其对进一步结构和功能研究的适用性。
