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

Nature Protocols volume 5, pages 574–587 (2010)Cite this article

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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 α-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.

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Figure 1: Canonical reaction steps of protein trans-splicing (PTS).
Figure 2: Outline of the in vivo segmental isotope-labeling procedure described in the text.
Figure 3: Plasmid maps of the vectors used for protein ligation by protein trans-splicing (PTS).
Figure 4: Demonstration of in vivo protein ligation by the dual expression.
Figure 5: The effect of the induction order and inducer concentrations on protein ligation of A and R domains from AlgE4 epimerase (A-R).
Figure 6: HSQC spectra from one example.

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Acknowledgements

We thank R. Wimmer, F.L. Aachmann and E. Buchinger for providing the plasmids for the AlgE4 experiments and S. Züger for Figure 1 . This work is supported by the grants from the Academy of Finland (118385), Sigrid Jusélius Foundation and the Biocentrum Helsinki.

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Authors and Affiliations

  1. Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

    Mikko Muona, A Sesilja Aranko, Vytas Raulinaitis & Hideo Iwaï

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Contributions

H.I. conceived and designed the experiments. M.M., A.S.A. and H.I. performed the experiments. M.M., A.S.A., V.R. and H.I. wrote the paper.

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Correspondence to Hideo Iwaï.

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Muona, M., Aranko, A., Raulinaitis, V. et al. Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro. Nat Protoc 5, 574–587 (2010). https://doi.org/10.1038/nprot.2009.240

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