Site-specific biotinylation of purified proteins using BirA

Methods Mol Biol. 2015:1266:171-84. doi: 10.1007/978-1-4939-2272-7_12.


The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / chemistry
  • Biotinylation
  • Carbon-Nitrogen Ligases / biosynthesis*
  • Carbon-Nitrogen Ligases / chemistry
  • Carbon-Nitrogen Ligases / isolation & purification
  • Chromatography, Affinity
  • Escherichia coli
  • Escherichia coli Proteins / biosynthesis*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / isolation & purification
  • Glutathione Transferase / biosynthesis
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / isolation & purification
  • Polymerase Chain Reaction
  • Protein Binding
  • Protein Engineering
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Repressor Proteins / biosynthesis*
  • Repressor Proteins / chemistry
  • Repressor Proteins / isolation & purification
  • Streptavidin / chemistry


  • Escherichia coli Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Biotin
  • Streptavidin
  • Glutathione Transferase
  • Carbon-Nitrogen Ligases
  • birA protein, E coli