title: Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.
abstract: A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.
authors.affiliation: Division of Oncology, Department of Medicine, Washington University School of Medicine, Campus Box 8076, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
authors.forename: Kwabena
authors.initials: K
authors.lastname: Sarpong
authors.affiliation: Division of Oncology, Department of Medicine, Washington University School of Medicine, Campus Box 8076, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Electronic address: rbose@wustl.edu.
authors.forename: Ron
authors.initials: R
authors.lastname: Bose
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C028841:erythrocyte membrane band 4.1 protein; D019881:Aminoacyltransferases; C120542:sortase A; C512478:EGFR protein, human; D066246:ErbB Receptors; D010450:Endopeptidases; C099488:TEV protease; D003546:Cysteine Endopeptidases
keywords: Affinity tag; LPXTG; Post-labeling purification; Sortase; TEV