Creative Biolabs provides a novel technique, proximity-dependent biotin identification approach, recently described as BioID, for screening and identification of protein-protein interactions in vivo. It takes advantage of the ability of a mutant form of biotin ligase to promiscuously biotinylate proximal proteins, no matter directly interacting or vicinal ones. This proximity ligation assay is playing a crucial role in mapping local protein interactomes network and understanding comprehensive cell functions.
The Brief Introduction of BioID
BioID, a unique and readily accessible method, allows detecting protein-protein interactions that occur in intact cells. Its mechanism is relying on a promiscuous biotin protein ligase, which is fused to a bait protein expressed in living cells and then biotinylated proximate endogenous proteins with excess biotin during a defined labeling period. This capability of biotinylation is irrespective of whether these interact directly or indirectly with the fusion protein of interest or merely located in the same subcellular neighborhood. Following biotin-affinity capture of streptavidin, biotinylated proteins can be selectively isolated and identified by mass spectrometry.
Fig.1 Diagram of basic BioID.
To date, BioID represents a promising new approach for kinds of cell types from diverse species not only in mammalian cells but also in cellular constituents, such as insoluble nuclear lamina and centrosome. It has become a powerful tool and notably applicable to study insoluble proteins, identify low affinity or transient interactions, and is amenable to temporal regulation.
The Background of BioID
The original guide of BioID is devised by a method named DamID, where the prokaryotic Dam methylase fused to the protein of interest monitors DNA-protein interaction in eukaryotes. Analogous to DamID, BioID protocols utilize BirA*, a mutant of prokaryotic Escherichia coli (E.coli) biotin ligase, to indiscriminately biotinylate proximal proteins. And BirA* is derived from a homologous enzyme BirA (R118G mutated), a 35-kD DNA-binding biotin protein ligase.
Biotinylation is a two-step reaction, where the biotin ligase makes use of biotin and ATP to produce a highly reactive biotinoyl-5′-AMP intermediate, which subsequently reacts with a specific lysine residue on the target protein, releasing AMP. In BioID, a wild-type biotin ligase BirA has stringent specificity for its substrate, but its adopted mutation, BirA*, affects biotin ligase activity (disordered loop) rendering it a promiscuous biotinylation enzyme and can covalently modify primary amines in the immediate vicinity of BirA*. For highly reactive and short-lived biotinoyl-5′-AMP, the zone of modification in BioID is thought to be only ~10 nm in labeling radius around the BirA*-tagged protein.
A Workflow of BioID
There are two main stages splitting out in a BioID process: (i) generate a BioID fusion protein vector for stable expression within a mammalian cell or desired cell line; (ii) utilize the stable cell line to proceed a large-scale BioID pull-down for validation of potential protein-interactors by mass spectrometry. The overall scheme of BioID steps in detail as follows:
Fig.2 Flowchart of the BioID.
a. Construct BioID expression vector ligating bait with BirA*
b. Stably express BioID fusion protein in cells
c. Induce biotinylation by incubating excess biotin
d. Perform cell lysis, protein denaturation under relatively harsh conditions
e. Isolate biotinylated proteins with streptavidin-sepharose by biotin-affinity capture
f. Identify candidate proteins using mass-spectrometry or immunoblot analysis
The Merits of BioID
It provides multiple advantages over traditional approaches for studying protein-protein interactions, including:
Significantly, it is worthy to note that BioID biotinylation is a mark of potential proximity and not an evidence for physical interactions. Thus, Creative Biolabs could also provide subsequent tests, like immunoprecipitation assays, to further validate proximity interactors identified by BioID and determine which reflect direct interactions in the physiological condition of cells.