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Freiburger, L. ; Sonntag, M. ; Hennig, J. ; Li, J.* ; Zou, P. ; Sattler, M.

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

J. Biomol. NMR 63, 1-8 (2015)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
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.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Multi-domain Proteins ; Protein Expression ; Protein Ligation ; Segmental Isotope Labeling ; Sortase A
ISSN (print) / ISBN 0925-2738
e-ISSN 1573-5001
Journal Journal of Biomolecular NMR
Quellenangaben Volume: 63, Issue: 1, Pages: 1-8 Article Number: , Supplement: ,
Publisher Springer
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Structural Biology (STB)