caa a22 4500 445324961 CHVBK 20180317142717.0 cr unu---uuuuu 170323e20110101xx s 000 0 eng 10.1007/s10858-010-9464-2 doi (NATIONALLICENCE)springer-10.1007/s10858-010-9464-2 Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy [Elektronische Daten] [Mary Refaei, Al Combs, Douglas Kojetin, John Cavanagh, Carol Caperelli, Mark Rance, Jennifer Sapitro, Pearl Tsang] 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. Springer Science+Business Media B.V., 2010 Segmental labeling nationallicence Sortase A nationallicence Protein ligation nationallicence MecA nationallicence Enzyme-mediated ligation nationallicence NMR nationallicence HSQC : Heteronuclear single quantum coherence nationallicence SDS-PAGE : Sodium dodecyl sulfate polyacrylamide gel electrophoresis nationallicence SEC : Size-exclusion chromatography nationallicence NOE : Nuclear Overhauser effect nationallicence MS : Mass spectrometry nationallicence Refaei Mary Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut Combs Al Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut Kojetin Douglas Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut Cavanagh John Department of Molecular and Structural Biochemistry, North Carolina State University, 27695, Raleigh, NC, USA aut Caperelli Carol College of Pharmacy, University of Cincinnati, 45267-0004, Cincinnati, USA aut Rance Mark Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut Sapitro Jennifer Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut Tsang Pearl Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut Journal of Biomolecular NMR Springer Netherlands 49/1(2011-01-01), 3-7 0925-2738 49:1<3 2011 49 10858 https://doi.org/10.1007/s10858-010-9464-2 text/html Onlinezugriff via DOI 1 brief-communication jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-010-9464-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Refaei Mary Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut NATIONALLICENCE 700 1- Combs Al Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut NATIONALLICENCE 700 1- Kojetin Douglas Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut NATIONALLICENCE 700 1- Cavanagh John Department of Molecular and Structural Biochemistry, North Carolina State University, 27695, Raleigh, NC, USA aut NATIONALLICENCE 700 1- Caperelli Carol College of Pharmacy, University of Cincinnati, 45267-0004, Cincinnati, USA aut NATIONALLICENCE 700 1- Rance Mark Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 45267-0524, Cincinnati, USA aut NATIONALLICENCE 700 1- Sapitro Jennifer Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut NATIONALLICENCE 700 1- Tsang Pearl Department of Chemistry, University of Cincinnati, 45221-0172, Cincinnati, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 49/1(2011-01-01), 3-7 0925-2738 49:1<3 2011 49 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer