Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Abstract

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.

Figure 1.

Streptavidin Western blot analyses of crude cell extracts. Wild type and mutant BirA proteins were expressed in wild type strain JM109 as described in Materials and Methods. The crude extracts were separated by 12% SDS-PAGE, and analyzed by Western blotting with streptavidin-AP. Lanes 5–8 contained 10-fold more extract protein than lanes 1–4. (Lanes 1,5) Wild type BirA; (lanes 2,6) G115S BirA; (lanes 3,7) R118G BirA; and (lanes 4,8) Δ1–34 BirA (the N-terminal deletion mutant). The arrows indicate the bands corresponding to BirA and BCCP.

Figure 2.

In vitro biotinylation with purified wild type and R118G proteins. In vitro biotinylation reactions were initiated by addition of purified wild type or R118G mutant BirA proteins (to a final concentration of 20 nM) to a reaction mixture containing a 2 μM final concentration of a specific substrate (apo BCCP) or a promiscuous acceptor (BSA or RNAse A) followed by incubation for 1 h at 37°C as described in Materials and Methods, followed by SDS-PAGE. Biotinylation of each protein was then analyzed by Western blotting with streptavidin-AP. (Lane 1) apo BCCP lacking BirA (note that this apo BCCP preparation was slightly contaminated with holo BCCP accounting for the bands in lanes 1 and 5); (lane 2) apo BCCP with wild type BirA; (lane 3) BSA with wild type BirA; (lane 4) RNAse A with wild type BirA; (lane 5) apo BCCP lacking R118G BirA; (lane 6) apo BCCP with R118G BirA; (lane 7) BSA with R118G BirA; (lane 8) RNAse A with R118G BirA. The arrows show the migration positions of BSA, BCCP, and RNase A.

Figure 3.

Time course of in vitro biotinylation of apo BCCP and RNAse A with the wild type and R118G proteins. The experiment was done as described in Figure 2 ▶ and in Materials and Methods. Lanes 1–5 contained the specific substrate apo-BCCP whereas lanes 6–10 contained a promiscuous acceptor, RNAse A. Samples were taken after various time intervals. These were 30 min (lanes 1,6), 1 h (lanes 2,7), 2 h (lanes 3,8), 6 h (lanes 4,9), and 24 h (lanes 5,10). Biotinylation was analyzed by Western blotting with streptavidin-AP. The arrows indicate the bands corresponding to BCCP and RNase A.

Figure 4.

Preparation and self-biotinylation of unbiotinylated BirA. (A) Wild type and R118G mutant BirA proteins were over expressed in either strain JM109 or in the bio strain BM4092 and purified as described in Materials and Methods. When strain BM4092 was used, the medium was depleted of biotin by addition of avidin to the 2XYT medium prior to induction of protein expression. An SDS-PAGE gel is shown. The left panel (lanes 1–4) was stained with Coomassie blue (1.4 mg protein per lane), whereas the right panel (lanes 5–8) was submitted to streptavidin-AP Western blot analysis (100 ng protein per lane). (Lanes 1,5) Wild type BirA expressed in JM109; (lanes 2,6) R118G BirA expressed in JM109; (lanes 3,7) wild type BirA expressed in BM4092; (lanes 4,8) R118G BirA expressed in BM4092. (B) The unbiotinylated preparations of the wild type (lanes 1–4) and R118G (lanes 5–8) proteins were used for in vitro self-biotinylation reactions (Materials and Methods). The BirA concentrations were varied as given on the figure. (Lanes 1,5) 250 nM; (lanes 2,6) 500 nM; (lanes 3,7) 750 nM; (lanes 4,8) 1 μM. After 1 h at 37°C a volume of each reaction corresponding to 2 pmole of protein was taken from of the various samples, run on SDS-PAGE, and analyzed by Western blotting with streptavidin-AP.

Figure 5.

Proximity-dependent biotinylation with R118G BirA. (A) His-tagged R118G BirA (final concentration 400 nM) was incubated in the reaction mixtures containing acceptor (Penta-HIS antibody) at a final concentration of 200 nM with or without the specific competitor (HIS-CAT, final concentration 2 μM) or with a promiscuous acceptor (either BSA or RNAse A, final concentration 2 μM) for 1 h at 37°C as described in Materials and Methods. The samples were then separated on an SDS-PAGE gel and analyzed by Western blot with streptavidin-AP. (Lane 1) His-tagged R118G BirA with omission of ATP; (lane 2) His-tagged R118G BirA plus Penta-HIS antibody; (lane 3) His-tagged R118G BirA plus Penta-HIS antibody preblocked with His-tagged CAT; (lane 4) His-tagged R118G BirA plus Penta-HIS antibody plus BSA; (lane 5) His-tagged R118G BirA plus Penta-HIS antibody plus RNAse A. (B) The reaction conditions were exactly the same as those of Figure 5A ▶, except that 0.25 μCi d-[carbonyl-¹⁴C]biotin was used as the biotin source. After the reaction mixtures were resolved in 12% SDS-PAGE, the gel was dried and exposed on X-ray film for one month at −80°C. The lanes are as in panel A. The arrows indicate BSA, the heavy (HC) and light (LC) chains of anti-Penta-HIS antibody, His-tagged BirA, His-tagged CAT (His-CAT), and RNase A. Note that the His-tagged CAT preparation contains a degradation product of the CAT protein in addition to the full-length protein.

Figure 6.

A depiction of proximity-dependent biotinylation. (A) A conjugate (or a fusion protein) composed of a mutant BirA and a target module (TM) of interest is exposed to a mixture of target (T) molecules. Some target molecules (T-1) bind the TM whereas others (T-2) fail to bind. (B) Upon addition of biotin and ATP the mutant BirA synthesizes bio-5′-AMP, which is depicted as a solid circle (the biotin moiety) linked to AMP (the shaded hexagon). (C) The localized cloud of bio-5′-AMP chemically bio-tinylates all of the proteins in the vicinity of BirA, T-1, TM, and BirA. The T-2 molecules fail to become biotinylated because most bio-5′-AMP molecules are hydrolyzed (shown as unlinked circles and hexagons) before they can reach the distant T-2 molecules. For simplicity, the process is shown in stages, although in practice, the interaction of TM and T-1, bio-5-AMP synthesis, and chemical biotinylation would proceed simultaneously.