Site-specific biotinylation of purified proteins using BirA

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.

Fig.1

Principle of BirA use. (A) Biotin ligase (BirA) reaction, covalently linking free biotin to the lysine of AviTag. (B) Advantage of labeling with BirA compared to labeling with amine-reactive biotin N-hydroxysuccinimide (NHS) esters, illustrated with regard to a Fab antibody fragment.

Fig. 2

Common applications of BirA biotinylation of purified proteins.

Fig. 3

Expression and purification of GST-BirA. 14% SDS-PAGE with Coomassie staining of samples of the lysate (Lys) and soluble fraction (Sol) of E. coli expressing GST-BirA and varying amounts of the protein preparation purified with glutathione-resin.

Fig. 4

Design of primers for AviTag insertion using the inverse PCR mutagenesis method.

Fig. 5

Testing the extent of biotinylation by SDS-PAGE gel-shift. Coomassie-stained SDS-PAGE of an antibody fragment (Fab0.35) with an AviTag on the C-terminus of both the heavy and light chains. The lanes represent non-biotinylated Fab (nb), biotinylated Fab, biotinylated Fab with streptavidin (SA), and streptavidin alone. Streptavidin has 4 binding sites and so may associate with 1 or 2 chains of the biotinylated target, but this does not affect the calculation of the depletion of the original target protein band.