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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.

Original publication

DOI

10.1007/978-1-4939-2272-7_12

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2015

Volume

1266

Pages

171 - 184

Keywords

Biotin, Biotinylation, Carbon-Nitrogen Ligases, Chromatography, Affinity, Escherichia coli, Escherichia coli Proteins, Glutathione Transferase, Polymerase Chain Reaction, Protein Binding, Protein Engineering, Recombinant Fusion Proteins, Repressor Proteins, Streptavidin