A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif Shuyan Xiao, a Xiaolin Zhao, a Carla V. Finkielstein b and Daniel G. S. Capelluto a * 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 glutathi- one S-transferase (GST) fusion protein using minimal media containing [ 15 N] 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 À80 °C. Purifica- tion 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. Keywords: detergent; disabled-2; isotopes; NMR; recombinant peptide; urea Introduction Peptides are involved in a variety of biological processes acting as antimicrobial macromolecules, enzyme inhibitors, and signal transduction triggers. Peptide purification methodologies have improved over the last few years by reducing the amount of time and effort required to obtain intact, biologically active peptides suitable for structural studies. Initially, peptide purification technologies (molecular mass <10, 000 Da) involved selective precipitations and adsorption of peptides along with ion-exchange and gel filtration chromatography, which led to low yields; more advanced technology involving the use of reversed-phase HPLC has led to higher yields [1]. Reversed-phased HPLC is carried out using sequential chromatography on columns containing different classes of silica-based packing materials, with octadecyl (C 18 ) columns being the most popular because they are suitable for the purification of hydrophilic peptides. For hydrophobic peptides, however, C 4 and diphenyl columns are recommended [1]. These columns use acetonitrile as a solvent because of its low viscosity, low absorbance at 214 nm (which is important for detecting the peptide bonds), and relatively high volatility. However, the HPLC technology has limitations, including low yield in the purification of extremely hydrophilic peptides, which show low retention in most of the HPLC-based columns; acetonitrile-mediated denaturation; and insolubility of polypeptides [2]. Although peptides can easily be produced by chemical synthesis, the large quantities required for structural or functional assays make this method cost-ineffective, resulting in their overexpression in a heterologous system as the default option. Although many peptides can be overexpressed as fusion proteins using Escherichia coli and other expression systems, the major limitation is their aggregation propensity, resulting in a low- solubility behavior. Expression, yield, stability, and solubility of peptides were successfully tackled when maltose-binding protein [3], E/K coils [4], and a combination of the B1 immunoglob- ulin domain of streptococcal protein G (GB1) and His tags were employed [5]. Antimicrobial peptides, which are ~50 amino acids in length, exhibit more unusual properties, such as being hydro- phobic with an excess of positive charges, making them difficult to isolate [6]. Thioredoxin, GB1, and glutathione S-transferase (GST) are the most popular carrier proteins for fusion expression with peptides [7,8]. The reason for their popularity resides in their ability to increase the solubility of fusion proteins, with thioredoxin being most favorable because of its small size (11.8 kDa) and because of its role as a chaperone in facilitating protein folding [9]. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technology that provides detailed information about the * Correspondence to: Daniel G. S. Capelluto, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA. E-mail: capellut@vt.edu a Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061, USA b Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061, USA Abbreviations: Dab2, Disabled-2; DPC, dodecylphosphocholine; HSQC, heteronuclear single quantum coherence; SBM, sulfatide-binding motifs peptide. J. Pept. Sci. 2014 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 9 October 2013 Revised: 17 November 2013 Accepted: 26 November 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/psc.2604 1