Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

Anal Biochem

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:

Published: March 2017

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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303137PMC
http://dx.doi.org/10.1016/j.ab.2017.01.008DOI Listing

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