BioID-Principles, Features and Applications

What is BioID Technique?

BioID (proximity-dependent biotin identification) is a proximity dependent labeling technology established by Roux et al in 2015 to study protein interactions. It can also be developed to study the interactions between proteins and RNA, and proteins and DNA.

How BioID technology works?

A recombinant expression vector of the target protein with biotin ligase (BirA) is first constructed and then transfected into cells for amplified expression. Biotin is added to the medium of the transfected cells. BirA activates biotin, so that all relevant proteins within 10 nm diameter around the target protein can be labeled by biotin.

In E. coli, the substrate recognized by the biotin ligase BirA (231 AA) is specific and can biotinylate only a specific segment of the amino acid sequence. If the 118 position of BirA is mutated (R118G, denoted as BirA*, also known as BioID1), the specificity of the substrate it recognizes is greatly reduced, allowing the protein it acts on to be biotinylated without having to carry a specific amino acid sequence.

When activated, biotin ligase can disrupt the active biotin molecule (biotinyl-5-AMP), separating it from the ligase and enabling it to react with free primary amines on exposed lysine residues in neighboring proteins, thereby covalently binding biotin to the lysine residues of the substrate protein. Biotinylation is a very rare modification in the cell. Therefore, isolation of biotinylated proteins by biotin-specific affinity purification followed by LC MS/MS analysis allows the identification of candidate proteins.

BioID (Varnaitė et al., 2016)

Features of BioID

Biotin is a naturally occurring metabolic enzyme cofactor that is active only when covalently linked to the enzyme by the action of a specific protein-biotin ligase. Any biotinylated substrate can bind tightly to protein affin and streptavidin.

The main advantages of BioID are as follows.

1. High sensitivity - The affinity between biotin and streptavidin (Kd=10-15 mol/L) is >1,000-fold higher than antibody-mediated interactions, allowing for more efficient and stable capture of protein complexes.

2. High specificity - This method avoids the use of antibodies, thereby significantly reducing non-specific binding. In addition, the extraordinary stability of biotin-streptavidin allows for rigorous purification to eliminate protein contamination.

3. Highly adaptable - Binding of streptavidin to biotin is rapid, specific, and can still occur under conditions where most other proteins have been denatured, such as high temperature or 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate (SDS). Biotinylated proteins can be efficiently purified directly from crude extracts in a one-step procedure, whereas the most commonly used affinity tags require many purification steps prior to the affinity binding step.

4. Cost-effective and reliable - To pull down proteins, the common practice is to purchase antibodies. However, antibodies are expensive and the quality of antibodies can vary from supplier to supplier and batch to batch, which is costly and time consuming.

BioID for Proteomic Research

BioID in subcellular structure research

The proteome and transcriptome of subcellular structures have traditionally been analyzed by immunoprecipitation (Co-immunoprecipitation, Co-IP) and biochemical separation. However, both methods require prior lysis of cells, which tends to lose low affinity with transient protein interactions. Also co-IP is limited by available antibodies, and biochemical isolation is often not done with complete purification. In addition, not all subcellular structures can be isolated. bioID technology has been developed and used to study subcellular structures such as nuclei, nuclear pore complexes, centrosomal cilia complexes, mitochondria, stress granules and processing bodies.

Biotinylation of Y-Nups in the context of the whole NPC defines a practical labeling radius (Kim et al., 2014)

BioID in virus-host interaction analysis

Upon virus entry into host cells, virus-host protein interactions are the main way in which the viral life cycle is regulated. The fusion of BioID with viral proteins, thus identifying key host factors contained in the microenvironment of the viral replicase/transcriptase complex, facilitates the study of relevant mechanisms. Proximity-dependent biotinylation can also be used to study the process of tumor progression associated with viruses or viral targeting of organelles, among others.

BioID in drug target discovery

RAS is an oncogene that is difficult to intervene with drugs. kovals⁃ki et al. combined BioID with CRISPR screening to identify a new set of functional RAS-associated proteins and defined mTORC2 as a direct RAS effector. Interfering with Ras-mTORC2 interactions can weaken RAS-dependent tumorigenesis in vivo, providing a possible option for the treatment of refractory oncogenes.

Proximity-dependent protein labeling (BioID) experimental workflow (Kovalski et al., 2019).

Interaction network of 150 proteins common to all Ras isoforms (Kovalski et al., 2019).

In addition, BioID is suitable for studying the interaction of insoluble proteins, a feature that is particularly useful when analyzing neurodegenerative diseases characterized by protein aggregation.