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.
Recombinant Disabled-2 sulfatide-binding motif (SBM) can be efficiently isolated in an intact and active form for NMR studies. The reported approach can be easily applied to the isolation of recombinant peptides for structural studies. The figure displays 1H, 15N-HSQC overlaid spectra of 15N-labeled Disabled-2 SBM in the absence (red) and presence of dodecylphosphocholine micelles (black). Addition of micelles improved resolution of the spectrum of the peptide, suggesting that this region is responsible for Disabled-2 membrane targeting.