Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Kita, Sapporo 001-0021, Japan.
Protein Eng Des Sel. 2013 Jun;26(6):409-16. doi: 10.1093/protein/gzt012. Epub 2013 Mar 22.
Recently, the hydrostatic pressure refolding method was reported as a practical tool for solubilizing and refolding proteins from inclusion bodies; however, there have been only a few applications for protein structural studies. Here, we report the successful applications of the hydrostatic pressure refolding method to refold proteins, including the MOE-2 tandem zinc-finger, the p62 PB1 domain, the GCN2 RWD domain, and the mTOR FRB domain. Moreover, the absence of aggregation and the correct folding of solubilized protein samples were evaluated with size exclusion chromatography and NMR experiments. The analyses of NMR spectra for MOE-2 tandem zinc-finger and GCN2 RWD further led to the determination of tertiary structures, which are consistent with those from soluble fractions. Overall, our results indicate that the hydrostatic pressure method is effective for preparing samples for NMR structural studies.
最近，静压折叠法被报道为一种从包涵体中溶解和重折叠蛋白质的实用工具；然而，它在蛋白质结构研究中的应用还很少。在这里，我们报告了静压折叠法成功应用于重折叠蛋白质，包括 MOE-2 串联锌指、p62 PB1 结构域、GCN2 RWD 结构域和 mTOR FRB 结构域。此外，通过尺寸排阻色谱和 NMR 实验评估了可溶蛋白样品无聚集和正确折叠的情况。MOE-2 串联锌指和 GCN2 RWD 的 NMR 谱分析进一步确定了与可溶部分一致的三级结构。总的来说，我们的结果表明静压法是一种有效的制备用于 NMR 结构研究样品的方法。
