Journal of Biomolecular NMR

, Volume 49, Issue 1, pp 3–7

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

  • Mary Anne Refaei
  • Al Combs
  • Douglas J. Kojetin
  • John Cavanagh
  • Carol Caperelli
  • Mark Rance
  • Jennifer Sapitro
  • Pearl Tsang
Communication

DOI: 10.1007/s10858-010-9464-2

Cite this article as:
Refaei, M.A., Combs, A., Kojetin, D.J. et al. J Biomol NMR (2011) 49: 3. doi:10.1007/s10858-010-9464-2

Abstract

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ~40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

Keywords

Segmental labeling Sortase A Protein ligation MecA Enzyme-mediated ligation NMR 

Abbreviations

HSQC

Heteronuclear single quantum coherence

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC

Size-exclusion chromatography

NOE

Nuclear Overhauser effect

MS

Mass spectrometry

Supplementary material

10858_2010_9464_MOESM1_ESM.pdf (3.3 mb)
Supplementary material 1 (PDF 3399 kb)

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Mary Anne Refaei
    • 1
    • 5
  • Al Combs
    • 2
  • Douglas J. Kojetin
    • 2
    • 6
  • John Cavanagh
    • 3
  • Carol Caperelli
    • 4
  • Mark Rance
    • 2
  • Jennifer Sapitro
    • 1
  • Pearl Tsang
    • 1
  1. 1.Department of ChemistryUniversity of CincinnatiCincinnatiUSA
  2. 2.Department of Molecular Genetics, Biochemistry and MicrobiologyUniversity of CincinnatiCincinnatiUSA
  3. 3.Department of Molecular and Structural BiochemistryNorth Carolina State UniversityRaleighUSA
  4. 4.College of PharmacyUniversity of CincinnatiCincinnatiUSA
  5. 5.Department of BiochemistryThe Ohio State UniversityColumbusUSA
  6. 6.Department of Molecular TherapeuticsThe Scripps Research Institute—Scripps FloridaJupiterUSA

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