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Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.
- Source :
-
Analytical biochemistry [Anal Biochem] 2017 Mar 15; Vol. 521, pp. 55-58. Date of Electronic Publication: 2017 Jan 11. - Publication Year :
- 2017
-
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.<br /> (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Subjects :
- Chromatography, Affinity methods
Cytoskeletal Proteins isolation & purification
Cytoskeletal Proteins metabolism
ErbB Receptors isolation & purification
ErbB Receptors metabolism
Humans
Membrane Proteins isolation & purification
Membrane Proteins metabolism
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Staphylococcus aureus enzymology
Aminoacyltransferases metabolism
Bacterial Proteins metabolism
Cysteine Endopeptidases metabolism
Cytoskeletal Proteins chemistry
Endopeptidases metabolism
ErbB Receptors chemistry
Membrane Proteins chemistry
Peptide Fragments metabolism
Recombinant Fusion Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0309
- Volume :
- 521
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 28088451
- Full Text :
- https://doi.org/10.1016/j.ab.2017.01.008