caa a22 4500 44532662X CHVBK 20180317142723.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s10989-011-9251-2 doi (NATIONALLICENCE)springer-10.1007/s10989-011-9251-2 Expression and Purification of Isotopically Enriched MHC Binding Immunogenic Peptides for NMR Studies [Elektronische Daten] [Sachin Patil, Lilly Saleena, Yong-Wah Kim, Edith Weber, Hermann von Grafenstein] Peptides are important naturally occurring ligands of MHC molecules. X-ray crystallographic studies have enabled extensive characterization of such peptide ligands. Yet structural and dynamic changes of these peptides in the MHC bound state are not well understood. These conformational transitions are key to understanding the function of MHC molecules and for the development of peptide-based therapeutics. Employing NMR for such studies can fill this gap but it requires the availability of peptides labeled with NMR-active nuclei. Here we report production of nine-mer MHC-binding peptides for use in high resolution NMR studies. The method utilizes a fusion protein approach of attaching the peptide to an easily expressed bacterial protein. The fusion protein construct design allows for rapid purification of the fusion protein and avoids chemical modification of the peptide as a result of the cleavage reaction. The methods developed here allow for rapid cloning of additional MHC binding peptides without significant molecular biology effort. 8-10mg of mature freeze dried peptides can be obtained from 1 liter of minimal media, sufficient for NMR experimentation. Six uniformly 15N-labeled peptides have been successfully expressed in bacteria and NMR spectra with the expected number of well-resolved signals were recorded. The results obtained here will make peptide-MHC complexes amenable to structural analysis which has not been possible previously. Springer Science+Business Media, LLC, 2011 Isotopic labeling nationallicence Peptide expression nationallicence NMR nationallicence CNBr cleavage nationallicence Fusion protein nationallicence Structural studies nationallicence MHC class I nationallicence HLA nationallicence GB1 : Immunoglobulin-binding domain of streptococcal protein G nationallicence TFA : Trifluoro acetic acid nationallicence HPLC : High performance liquid chromatography nationallicence CNBr : Cyanogen bromide nationallicence ESI-MS : Electrospray ionization mass spectrometry nationallicence HSQC : Heteronuclear single quantum coherence nationallicence NOESY : Nuclear overhouser effect spectroscopy nationallicence TOCSY : Total correlation spectroscopy nationallicence Patil Sachin Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut Saleena Lilly Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut Kim Yong-Wah University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut Weber Edith Institute of Pharmaceutical Chemistry, University of Szeged, POB. 121, 6701, Szeged, Hungary aut von Grafenstein Hermann Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut International Journal of Peptide Research and Therapeutics Springer Netherlands 17/2(2011-06-01), 137-145 1573-3149 17:2<137 2011 17 10989 https://doi.org/10.1007/s10989-011-9251-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10989-011-9251-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Patil Sachin Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut NATIONALLICENCE 700 1- Saleena Lilly Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut NATIONALLICENCE 700 1- Kim Yong-Wah University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut NATIONALLICENCE 700 1- Weber Edith Institute of Pharmaceutical Chemistry, University of Szeged, POB. 121, 6701, Szeged, Hungary aut NATIONALLICENCE 700 1- von Grafenstein Hermann Department of Medicinal and Biological Chemistry, University of Toledo, 2801 W. Bancroft St., 43606, Toledo, OH, USA aut NATIONALLICENCE 773 0- International Journal of Peptide Research and Therapeutics Springer Netherlands 17/2(2011-06-01), 137-145 1573-3149 17:2<137 2011 17 10989 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer