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

- TEV protease cleaved proteins are amenable to sortase-mediated labeling.
- Post-labeling purification using an affinity tag in the peptide substrate results in >95% purity of the labeled protein.
- We demonstrate site-specific labeling of EGFR tyrosine kinase and the nanodisc protein, MSP.

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.