Site-specific biotinylation of purified proteins using BirA

E. coli biotin ligase (BirA) covalently attaches free biotin to the lysine of the 15-amino-acid AviTag peptide (GLNDIFEAQKIEWHE), giving a homogeneous biotinylated product with high yield. Chemical biotinylation with amine-reactive biotin N-hydroxysuccinimide (NHS) esters often generates heterogeneous products that may have impaired function.

The natural E. coli BirA substrate, Biotin Carboxyl Carrier Protein (BCCP), requires fusion of at least 75 residues to the target protein. The 15-amino-acid AviTag (acceptor peptide, AP), obtained by phage display selection, is a superior BirA substrate to BCCP while being much shorter, which extends the range of protein sites amenable to site-specific enzymatic biotinylation.

The minimal BirA substrate peptide is a 13-residue sequence (LX§IFEAQKIEWR, where X is any residue and § is any residue except L, V, I, W, F or Y), which was further optimized for biotinylation rate to give AviTag (GLNDIFEAQKIEWHE). Related BirA substrate peptides are BioTag (ALNDIFEAQKIEWHA, 15 residues), BLRP (MAGGLNDIFEAQKIEWHEDTGGS, 23 residues, containing the AviTag core) and the 15-residue BirA Substrate Peptide BSP (LHHILDAQKMVWNHR).

AviTag works at either the N-terminus or the C-terminus of a target protein and can also be inserted into protein loops. BirA-mediated biotinylation after AviTag insertion into loops succeeds in the E. coli flagellar hook, the Cystic Fibrosis Transmembrane Regulator (CFTR) and Dicer. For Dicer, insertion in short loops that are disordered in the crystal structure or less highly conserved is advised, because only some insertion sites remain functional.

The Lys-to-Ala mutant of AviTag (GLNDIFEAQAIEWHE) is not biotinylated by BirA and serves as an effective negative-control peptide.

A flexible two-residue GS linker between AviTag and the target protein, or any other surrounding peptide tag or domain, is recommended. If N-terminal or C-terminal AviTag constructs are not biotinylated or give low protein yield, increasing the spacer to 6 residues is the first remedy, followed by switching to the BSP peptide.

AviTag fusions expressed in E. coli can be partly biotinylated by the cell's own BirA, but this reaction often does not reach completion, even in a strain with BirA overexpressed (AVB101). Adding purified BirA to an AviTag fusion in the absence of ATP or biotin can still produce some biotinylation, because biotin-AMP is pre-bound to the purified protein.

In vitro biotinylation of an AviTag-fused protein with GST-BirA is run in PBS with MgCl2, ATP and D-biotin for 1 hour at 30 °C with gentle mixing, followed by a second 1-hour incubation after adding the same amount of fresh biotin and GST-BirA. Biotinylation in PBS is reported to be quantitative, although an AviTag-fusion concentration above 40 µM is preferable because lower concentrations make biotinylation less efficient.

BirA biotinylation of a target protein is generally irreversible in vitro, so an apparent loss of biotinylation most likely reflects proteolysis that separates the biotinylation site from the rest of the target protein.

GST-BirA, His6-BirA and MBP-BirA fusions all express well in E. coli, and GST-BirA can be efficiently removed from the biotinylated substrate after the reaction by passing the mixture through glutathione-agarose. GST-BirA purified from E. coli on glutathione resin yields 10-20 mg per litre of expression culture, has an ε280 of 90,550 M−1 cm−1, and concentrations much greater than 50 µM may crash out of solution.

The extent of AviTag biotinylation is quantified by an SDS-PAGE streptavidin gel-shift. The biotinylated protein is boiled in SDS sample buffer and cooled, then a 2- to 5-fold molar excess of streptavidin is added and incubated for 5 minutes at room temperature. Provided the gel does not get excessively warm, streptavidin stays a tetramer bound to biotin conjugates in SDS-PAGE, and the loss of the target band relative to a no-streptavidin lane is measured by densitometry. The streptavidin tetramer runs at 50-60 kDa and is visible on 10, 12, 14 and 16% gels.

A band of free streptavidin in the lane containing biotinylated protein plus streptavidin verifies that streptavidin was in excess and that all biotinylated protein was bound. Streptavidin may increase in mobility upon binding a biotin conjugate, depending on the size and charge of the conjugate.

Streptavidin binds biotin with a Kd of 4 × 10−14 M, and binding still occurs under conditions where most other proteins denature, such as high temperatures, 6 M guanidinium hydrochloride or 1% SDS. Avidin is not recommended for capturing biotinylated proteins because its positive charge promotes non-specific binding to cells and DNA, whereas neutravidin should be satisfactory for many applications.

BirA from E. coli or other species can ligate biotin analogs to the peptide tag, including desthiobiotin for reversible streptavidin binding and analogs bearing keto, azido or alkyne groups for bio-orthogonal reactions. Only small changes to the structure of biotin are tolerated by biotin ligase, so the related ligase LplA is more amenable to direct incorporation of fluorophores.

BirA biotinylates AviTag-type substrate peptides specifically in the cytosol, in the secretory pathway and at the cell surface in mammalian and invertebrate systems. Targeting BirA to a specific chromatin-associated protein biotinylates AviTag-linked nucleosome populations, and targeting BirA to one synaptic membrane biotinylates AviTag-proteins on the opposite synaptic membrane.

Expressing a BirA-substrate peptide on a nuclear envelope protein together with BirA in specific tissues of Arabidopsis thaliana, Caenorhabditis elegans or Drosophila melanogaster allows nuclei from specific cell types to be isolated by streptavidin pull-down.

Monovalent streptavidin (mSA) allows precise control over the assembly of biotin conjugates, and the streptavidin variant traptavidin has a 10-fold lower off-rate for biotin and enhanced thermal stability.

Covalent peptide-tag systems based on split inteins, sortase and SpyCatcher have not yet matched the sensitivity of detection of streptavidin or avidin, and a key future development would be to raise the affinity of monomeric streptavidin to that of tetrameric streptavidin.

An AviTag can be inserted at a chosen site in a plasmid by inverse PCR mutagenesis without requiring restriction sites nearby. The forward and reverse primers each carry 18-25 bp matching the parental sequence with a calculated annealing temperature of at least 55 °C, amplification uses 12 cycles with KOD hot start polymerase, and the product is treated with DpnI and then ligated with T4 polynucleotide kinase and T4 DNA ligase before transformation into E. coli.