Avi biotinylation tag

Fifteen-residue peptide, GLNDIFEAQKIEWHE, that the E. coli biotin ligase BirA attaches biotin to at a single lysine. A tagged protein is then captured on streptavidin, giving site-specific biotinylation without chemical labeling.

Length: 45 bp (15 aa)

Uses: labeling, purification

Cleavable: No

Detected by: streptavidin

Origin: Peptide-library screen for BirA substrates, then optimized

Characteristics

BirA transfers biotin to the lysine at position 10 of GLNDIFEAQKIEWHE (15 amino acids, 45 nucleotides). The sequence bears little resemblance to the natural BirA substrate BCCP, which needs at least 75 fused residues. A 14-residue core is the minimal substrate, with kcat/Km 10,000 ± 500 M-1 s-1 versus 11,900 ± 400 M-1 s-1 for BCCP87 (stopped-flow, 20 °C, pH 7.5). The 15-mer is recommended for fusions for its consistently higher rate. It works at the N or C terminus or in exposed loops, though internal sites give variable results depending on how the tag folds; the Lys to Ala mutant is not biotinylated. In vitro, 38 µM AviTag fusion is over 95% biotinylated in 30 min at 30 °C with 25 µg/ml BirA.

Applications: Site-specific biotinylation of purified AviTag fusions for MHC class I tetramerization with streptavidin, immobilization on chromatography columns, chips, atomic force microscope tips and nanoparticles, and detection of cells with streptavidin conjugates. BirA also biotinylates the tag in the cytosol, secretory pathway and cell surface of mammalian and invertebrate systems.

Limitations: Requires BirA, ATP, MgCl2 and biotin. With the same BirA amount, 40 µM substrate is biotinylated in about 30 min but 4 µM takes about 5 h. In E. coli with endogenous BirA, fusions are biotinylated about 10-30% at low or moderate expression and 2-6% when highly overexpressed. Biotinylation is generally irreversible in vitro, and streptavidin binds biotin with Kd 4 × 10-14 M and survives 1% SDS.

Sequence

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References

  1. Beckett et al. (1999). A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci - Beckett 1999 AviTag Substrate
  2. Fairhead et al. (2014). Site-specific biotinylation of purified proteins using BirA. Methods Mol Biol - Fairhead 2015 BirA Biotinylation
  3. Cull et al. (2000). Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol - Cull 2000 Biotinylation Peptide Tags