Gronenborn A M, Clore G M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Protein Sci. 1996 Jan;5(1):174-7. doi: 10.1002/pro.5560050123.
A simple and rapid method based on 15N labeling and 1H-15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin-binding domain of streptococcal protein G (56 residues) and human interleukin-1 beta (153 residues). It is shown that high quality 1H-15N correlation spectra, recorded in as little as 15 min and displaying only cross-peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.
本文提出了一种基于15N标记和1H-15N异核单量子相干光谱的简单快速方法，可直接评估大肠杆菌粗提物中过表达蛋白质的结构完整性，无需任何纯化。该方法通过两种不同的表达系统和两种不同的蛋白质进行了验证，即链球菌蛋白G的B1免疫球蛋白结合结构域（56个残基）和人白细胞介素-1β（153个残基）。结果表明，从大肠杆菌粗提物中可以获得高质量的1H-15N相关光谱，记录时间短至15分钟，且仅显示来自目标过表达蛋白质的交叉峰。
