A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif

Authors

  • Shuyan Xiao,

    1. Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
    Search for more papers by this author
  • Xiaolin Zhao,

    1. Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
    Search for more papers by this author
  • Carla V. Finkielstein,

    1. Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
    Search for more papers by this author
  • Daniel G. S. Capelluto

    Corresponding author
    1. Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
    • Correspondence to: Daniel G. S. Capelluto, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA. E-mail: capellut@vt.edu

    Search for more papers by this author

Abstract

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.

Ancillary