Tang Meng, Cao Ruiyu, Du Lingyu, Xu Jikang, Wu Bin, OuYang Bo
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Chembiochem. 2022 Jan 19;23(2):e202100514. doi: 10.1002/cbic.202100514. Epub 2021 Dec 9.
In addition to a membrane anchor, the transmembrane domain (TMD) of single-pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross-membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence-specific nickel-assisted cleavage (SNAC)-tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike-TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high-quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.
除了膜锚定结构外，单通道跨膜蛋白（SPTMPs）的跨膜结构域（TMD）最近在跨膜活性或受体组装/聚集过程中显示出重要作用。然而，这些小的TMD肽通常具有疏水性且动态性强，难以表达和纯化。在此，我们整合了TrpLE融合蛋白和序列特异性镍辅助切割（SNAC）标签的优势，在温和条件下高效生产小的TMD肽，该方法使用镍作为切割试剂，避免了使用有毒的溴化氰（CNBr）。此外，该方法简化了下游蛋白质的纯化和重组过程。包括严重急性呼吸综合征冠状病毒2（SARS2）的刺突跨膜结构域（Spike-TMD）在内的两个代表性TMD，成功获得了高质量的核磁共振（NMR）谱图。因此，我们的研究为小跨膜蛋白的一般结构表征提供了一种更高效、实用 的方法。
