A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif.
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.
Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, 24061, USA.
Shuyan
S
Xiao
Xiaolin
X
Zhao
Carla V
CV
Finkielstein
Daniel G S
DG
Capelluto
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D007554:Isotopes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C102885:DAB2 protein, human; D007554:Isotopes; D010455:Peptides; D025521:Tumor Suppressor Proteins
NMR; detergent; disabled-2; isotopes; recombinant peptide; urea