Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Nat Protoc. 2010 Mar;5(3):574-87. doi: 10.1038/nprot.2009.240. Epub 2010 Mar 4.
Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.
分段同位素标记是一种强大的标记技术，可用于减少核磁共振（NMR）信号重叠，该技术通过将稳定同位素仅掺入蛋白质的一个区域中进行 NMR 检测，从而与较大的蛋白质相关联。分段同位素标记不仅可以降低 NMR 光谱的复杂性，而且还可以通过三共振 NMR 实验保留进行顺序共振分配的可能性。我们描述了通过使用无任何重折叠步骤或α-硫代酯修饰的分裂 DnaE 内含肽通过蛋白质转剪接（PTS）在体内（即在大肠杆菌中）和体外对多结构域和融合蛋白进行分段同位素标记的方案。PTS 方法的优点在于，它可以通过带有两个可控启动子的延时双重表达系统在体内进行。一旦构建了所需的载体，分段同位素标记的蛋白质可以在大肠杆菌中在 1 天内表达。根据使用的方案，分段标记样品的总制备时间可以短至 7-13 天。
