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Journal of Biomolecular NMR

, Volume 63, Issue 1, pp 1–8 | Cite as

Efficient segmental isotope labeling of multi-domain proteins using Sortase A

  • Lee Freiburger
  • Miriam Sonntag
  • Janosch Hennig
  • Jian Li
  • Peijian Zou
  • Michael Sattler
Communication

Abstract

NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.

Keywords

Protein ligation Sortase A Multi-domain proteins Segmental isotope labeling Protein expression 

Notes

Acknowledgments

We thank Iren Wang for discussions and TIA-1 expression protocols and Arie Geerlof at the Protein Expression and Purification Facility of the Helmholtz Center Munich for discussion. This work was supported by The Deutsche Forschungsgemeinschaft (DFG, SFB1035 and GRK1721 to M.S.), the Center for Integrated Protein Science Munich (CIPSM). L.F. acknowledges support by an European Molecular Biology Organization (EMBO) Longterm Fellowship and Marie Curie FP7 International Incoming Fellowship. J. H. acknowledges the EMBO for a long-term fellowship (ALTF-276-2010) and the Swedish Research Council (Vetenskapsrådet) for a postdoc fellowship.

Supplementary material

10858_2015_9981_MOESM1_ESM.pdf (959 kb)
Supplementary material 1 (PDF 959 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Lee Freiburger
    • 1
    • 2
  • Miriam Sonntag
    • 1
    • 2
  • Janosch Hennig
    • 1
    • 2
  • Jian Li
    • 3
  • Peijian Zou
    • 1
    • 2
    • 3
  • Michael Sattler
    • 1
    • 2
    • 3
  1. 1.Institute of Structural BiologyHelmholtz Zentrum MünchenNeuherbergGermany
  2. 2.Center for Integrated Protein Science Munich (CIPSM) at Department of ChemistryTechnische Universität MünchenGarchingGermany
  3. 3.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina

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