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Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Nature Biotechnology volume 23, pages 736–740 (2005)Cite this article

Abstract

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins1,2,3. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.

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Figure 1: Biosynthetic preparation of segmentally isotope-labeled proteins using trans-protein splicing.
Figure 2: Structure of the two plasmids for in vivo segmental isotopic labeling.
Figure 3: SDS-PAGE analysis of in vivo protein ligation.
Figure 4: NMR spectra of the fully and segmentally isotope-labeled proteins.

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Acknowledgements

We thank Henry Paulus, Hideki Taguchi and Stephen F. Marino for providing the plasmids containing Ssp DnaE intein, Sup35NM and GB1, respectively. We also thank Pui Hang Tam, Jennifer Jin, Sarah Figley for their technical assistance and the Saskatchewan Structural Science Center for the use of NMR instruments. H.I. thanks Peter Güntert for his continuous support. This study is supported by the National Science and Engineering Research Council of Canada (298493-4) and the Biomolecular Structure Teaching and Research Program at the University of Saskatchewan.

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Authors and Affiliations

  1. Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, S7N 5C9, Saskatchewan, Canada

    Sara Züger & Hideo Iwai

  2. Department of Biochemistry, University of Saskatchewan, 110 Science Place, Saskatoon, S7N 5C9, Saskatchewan, Canada

    Hideo Iwai

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  1. Sara Züger
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Correspondence to Hideo Iwai.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Strips from the HNCACB spectrum of [13C, 15N]-GB1-CBD (PDF 260 kb)

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Züger, S., Iwai, H. Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nat Biotechnol 23, 736–740 (2005). https://doi.org/10.1038/nbt1097

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