Site-Specific Biotinylation of Purified Proteins Using BirA

We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDSPAGE to quantify the extent of biotinylation

Michael Fairhead; Mark Howarth

2015

Scholarcy highlights

  • Biotin is a cofactor for carboxylase enzymes, present in all living organisms
  • Phage display selection enabled the development of the AviTag, which is superior to Biotin Carboxyl Carrier Protein as a BirA substrate but only 15 amino acids in length, so extending the range of protein sites amenable to site-specific enzymatic biotinylation
  • Sensitive detection - an in vitro biotinylated protein can be added to cells and subsequently recognized with high affinity by streptavidin conjugates
  • The methods described below utilize a glutathione-S-transferase-BirA fusion protein, but are adaptable to His6-tagged or Maltose Binding Protein fusion constructs
  • We suggest using a modified inverse PCR mutagenesis or Site-directed Ligase-Independent Mutagenesis reaction, which enables the insertion of the substrate peptide without requiring any restriction sites nearby
  • Provided the gel does not get excessively warm during the run, streptavidin will retain its native tetramer structure and remain bound to biotin conjugates under normal sodium dodecyl sulfate-PAGE conditions
  • We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDSPAGE to quantify the extent of biotinylation

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