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.

Original languageEnglish
Pages (from-to)55-58
Number of pages4
JournalAnalytical Biochemistry
Volume521
DOIs
StatePublished - Mar 15 2017

Keywords

  • Affinity tag
  • LPXTG
  • Post-labeling purification
  • Sortase
  • TEV

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