TurboID

Proximity-based labeling technique makes great contributions to protein-protein interaction detection and protein interactome network mapping. As an experienced services provider in pre-clinical research and drug discovery, Creative Biolabs has won a good reputation in completing the identification of protein-protein interactions. Based on the existing proximity-dependent biotin identification (BioID) service, our professional scientists here provides new TurboID services for protein-protein interaction exploration in vivo.

The Background of TurboID

Protein-protein interactions (PPIs) play an essential role in most cellular and biological processes, such as DNA replication, signal molecule transmission, and immune defense. A variety of in vivo and in vitro methods have been developed to identify PPIs, including commonly used yeast two-hybrid screening, co-immunoprecipitation, pull-down, protein microarrays, etc. A novel technique enzyme-catalyzed proximity labeling (PL) has emerged recently, which has been popularly and valuably utilized for the PPIs' identification, especially for the protein interactome mapping.

PL assay is performed by labeling a protein of interest with an enzyme that can promiscuously biotinylate biomolecules in a proximity-dependent manner, and then identify PPIs through methods of affinity purification and mass spectrometry (MS). Several test systems have been developed so far, among which Ascorbate Peroxidase (APEX)-based PL and Biotin ligase-based PL are the most commonly used approaches in PPI identification. And TurboID is a recently developed technique based on proximity-dependent biotin identification (BioID), a convenient PPI detection method using a promiscuous mutant E. coli biotin ligase.

Fig.1 Schematic representation of proximity-labeling systems. (Yang, 2020)

TurboID, an Advanced Version of BioID

BioID is a simple and non-toxic method to identify PPIs that developed based on proximal protein biotin labeling by prokaryotic E. coli biotin ligase BirA mutant BirA. BirA/BirA ligase is a highly conserved enzyme that catalyzes the biotin and adenosine triphosphate (ATP) to produce a reactive intermediate, biotin–adenosine monophosphate (biotin-5ʹ-AMP). The wild-type BirA shows high affinity and specificity for biotin-5ʹ-AMP, while the mutant BirA (R118G) has a remarkably reduced affinity to biotin-5ʹ-AMP. The reactive biotin-5ʹ-AMP is short-lived and can diffuse from its active site and indiscriminately biotinylate the lysine residues of proteins nearby the BirA (about within a radius of 10 nm), that is proximity labeling. The labeled proteins then are isolated and purified by avidin purification, and identified by MS.

Since the advent of BioID, it has widely been applied PPI detection in mammalian cells, mice, parasites, yeast, and other live cells. However, BioID has not achieved the expected result due to long labeling time (about 18-24 h), background interference of biotin and unstable biotin absorption efficiency in the plant. Thus, improved BioID methods have been developed to over these limitations, such as biotin ligase BioID2 from Aquifex aeolicus, biotin ligase BASU from the Bacillus subtilis, and engineered biotin ligase TurboID from yeast display.

Fig.2 BioID workflow for the identification of direct and proximal protein-protein interactions. (Cheerathodi, 2020)

TurboID and Split-TurboID

Through the yeast surface display directed evolution, scientists have engineered BirA to produce two promiscuous biotin ligases: a 35 kD TurboID and a 28 kD miniTurbo (Figure 3), both of which are non-toxic and more active than previous BioID. Compare with current BioID, TurboID and miniTurbo also show advantages in:

1. Shorter labeling time. Both TurboID and miniTurbo require 10 min or less (much faster than ~18 h of BioID) to biotinylate adjacent proteins, enabling to detect dynamic biological processes with higher temporal resolution.
2. Temperature adaptability. Both TurboID and miniTurbo also can maintain efficient catalysis activity at lower temperatures (25°C), which overcomes limitations (required 37°C) of BioID and APEX systems applied to plants, worms, yeast, etc.
3. Lower labeling background. Since miniTurbo has a lower affinity for biotin than TurboID, it can reduce the labeling background and minimize the adverse effect of label fusion on the function of the target protein.

Fig.3 E. coli biotin ligase structure with sites mutated in TurboID (left) and miniTurbo (right) shown in red. (Branon, 2018)

Based on TurboID, split-TurboID is a derivative method similar to bimolecular fluorescence complementation assay, which exhibits higher targeting specificity than full-length TurboID. The TurboID ligase is split into two inactive fragments and respectively fused with the target proteins, by which TurboID ligase can be reconstituted and restore catalytic activity through target proteins' interactions. The proximal interacted proteins are biotinylated after adding biotin into the system, and then these biotinylated proteins by TurboID or split-TurboID are purified and identified like BioID workflow.

Fig.4 Proximity-dependent biotinylation catalyzed by TurboID and split-TurboID. (Cho, 2020)

Let's Work Together to Fulfil Your PPI Identification!

As a recognized biotech company in the field of drug discovery, Creative Biolabs has accumulated abundant expertise and experience in protein-protein interaction identifications. Based on the powerful technology platform, we are professional in providing one-stop and tailored solutions to advance the PPI detection for our valued customers. In addition to our seasoned yeast two-hybrid (Y2H) and bacterial two-hybrid (B2H) services, here we successively launched BioID and TurboID to facilitate protein interaction research.

Just feel free to contact us and communicate with us about your questions, our experienced technicians will give you the most tailored solutions to you.

References

1. Yang, X.X; et al. Proximity labeling: an emerging tool for probing in planta molecular interactions. Plant Communications. 2020, 2(2): 100137.
2. Cheerathodi, M.R.; and Meckes, D. BioID Combined with Mass Spectrometry to Study Herpesvirus Protein–Protein Interaction Networks. In book: Herpes Simplex Virus. Methods in molecular biology (Clifton, N.J.). 2060, pp: 327-341.
3. Branon, T.C.; et al. Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology. 2018, 36: 880-887.
4. Cho, K.F.; et al. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nature Protocols. 2020, 15: 3971-3999.

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