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

Title data

Freiburger, Lee ; Sonntag, Miriam ; Hennig, Janosch ; Li, Jian ; Zou, Peijian ; Sattler, Michael:
Efficient segmental isotope labeling of multi-domain proteins using Sortase A.
In: Journal of Biomolecular NMR. Vol. 63 (2015) Issue 1 . - pp. 1-8.
ISSN 1573-5001
DOI: https://doi.org/10.1007/s10858-015-9981-0

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Keywords: Multi-domain proteins; Protein expression; Protein ligation; Segmental isotope labeling; Sortase A
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry - Univ.-Prof. Dr. Janosch Hennig
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 07 Oct 2021 12:44
Last Modified: 07 Oct 2021 12:44
URI: https://eref.uni-bayreuth.de/id/eprint/67238