A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif
Article first published online: 27 JAN 2014
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Journal of Peptide Science
Volume 20, Issue 3, pages 216–222, March 2014
How to Cite
Xiao, S., Zhao, X., Finkielstein, C. V. and Capelluto, D. G. S. (2014), A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif. J. Peptide Sci., 20: 216–222. doi: 10.1002/psc.2604
- Issue published online: 11 FEB 2014
- Article first published online: 27 JAN 2014
- Manuscript Accepted: 26 NOV 2013
- Manuscript Revised: 17 NOV 2013
- Manuscript Received: 9 OCT 2013
- the National Science Foundation CAREER. Grant Number: MCB-0844491
- the American Heart Association. Grant Number: 11BGIA6160000
- recombinant peptide;
A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [15N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at −80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.