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Site-Specific Biotinylation of Purified Proteins Using BirA

  • Michael Fairhead
  • Mark HowarthEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)

Abstract

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.

Key words

Neutravidin Streptavidin-biotin Femtomolar Nanotechnology Bionanotechnology 

Notes

Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC). We thank Jayati Jain (Howarth laboratory) for providing Fig. 5.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK

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