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Nature Research, Nature Protocols, 3(5), p. 574-587

DOI: 10.1038/nprot.2009.240

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Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro

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This paper is available in a repository.

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Abstract

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.