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


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Article Info

DATE

2015-09

AUTHORS

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

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. More... »

PAGES

1-8

References to SciGraph publications

  • 2014-11. Structural basis for the assembly of the Sxl–Unr translation regulatory complex in NATURE
  • 2011-01. Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy in JOURNAL OF BIOMOLECULAR NMR
  • 2009-03. Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method in JOURNAL OF BIOMOLECULAR NMR
  • 2013-05. Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex in JOURNAL OF BIOMOLECULAR NMR
  • 2008-09. Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A in BIOTECHNOLOGY LETTERS
  • 2010-03. Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro in NATURE PROTOCOLS
  • 2007-09. Solution NMR of supramolecular complexes: providing new insights into function in NATURE METHODS
  • 2010-01. Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins in JOURNAL OF BIOMOLECULAR NMR
  • 2013-09. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions in NATURE PROTOCOLS
  • 2006-08. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy in NATURE PROTOCOLS
  • 2013-10-13. The structure of the box C/D enzyme reveals regulation of RNA methylation in NATURE
  • 2005-06. Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies in NATURE BIOTECHNOLOGY
  • 2014-02. Depsipeptide substrates for sortase-mediated N-terminal protein ligation in NATURE PROTOCOLS
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s10858-015-9981-0

    DOI

    http://dx.doi.org/10.1007/s10858-015-9981-0

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1004886800

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/26319988


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