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.

Other optional protein-protein interaction (PPI) assay services:

• Two Hybrid Services
• Protein-Fragment Complementation Assay (PCA) Service
• Proximity-Dependent Biotin Identification (BioID) Service
• Protein-Protein Interaction Assay by X-Ray Crystallography Service
• Mammalian Protein-Protein Interaction Trap (MAPPIT) Service
• Magic™ Conversable Mammalian Library (CML) Platform for Biomolecular Interaction Discovery
• Y2H-Based Drug-Target Interaction Identification
• Yeast Three-Hybrid (Y3H) for Protein Interaction Identification

Published Data

• TurboID-Based Proximity Labeling Captures the Substrates of Kinases and Phosphatases

Fig. 5 TurboID-based identification of BIN2-proximal proteins in Arabidopsis. (Tae-Wuk Kim, 2023)

Since enzyme-substrate interactions are often short-lived, the enzyme-substrate relationship in post-translational modification (PTM) networks is often difficult to explain. Here, the researchers demonstrated that TurboID-based proximity labeling (TbPL) can effectively and specifically capture kinase and phosphatase substrates. More than 400 proximal proteins of the Arabidopsis thaliana BIN2 protein were identified by TbPL-mass spectrometry. Most of the proximal proteins of BIN2 show BIN2-dependent phosphorylation in vivo or in vitro. Protein-protein interaction network analysis showed that the proximal proteins of BIN2 included interactions with BIN2 substrates, which revealed a high level of interaction between BIN2 proximal proteins. The researchers established a BIN2 signaling network through proteomic analysis and revealed the role of BIN2 in regulating key cellular processes.

• Phase-Separated CO2-Fixing Pyrenoid Proteome Determined by TurboID

Fig. 6 Establishment and optimization of TurboID labeling in the Chlamydomonas chloroplast using RBCS2-TurboID lines. (Chun Sing Lau, 2023)

Phase separation is the basis of many biologically important cellular events, such as RNA metabolism, signal transduction and CO2 fixation. Traditional proteomics techniques limit the understanding and application of phase separation. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, the researchers developed a proximal labeling technique based on TurboID, in which the proximal proteins in Chlamydomonas chloroplasts can be labeled by biotin free radicals produced by TurboID labeling proteins. Then, by fusing the two core protein nuclear components with the TurboID tag, the scientists generated a nuclear proximal group containing most of the known nuclear proteins as well as new nuclear candidates. Fluorescent protein labeling of 7 previously uncharacterized TurboID recognition proteins showed that 6 of them were located in a series of subpyrenoid regions. The resulting proxiome also suggests that nuclear proteins have new secondary functions in RNA-related processes and redox-sensitive iron-sulfur cluster metabolism.

FAQ

1. What is TurboID and how does it work?

   TurboID is an engineered biotin ligase that is used for proximity labeling in biological research. It is designed to be a faster and more efficient version of the protein labeling enzyme BioID. TurboID catalyzes the biotinylation of proteins that are near it in living cells and tissues. This process involves the enzyme facilitating the addition of a biotin tag to lysine residues on proteins that are within approximately 10 nanometers of TurboID. This proximity labeling is particularly useful for studying protein interactions and the composition of protein complexes in their native cellular environment. The enhanced speed and efficiency of TurboID allow for shorter labeling times, ranging from minutes to a few hours, which is significantly faster compared to the original BioID that may require up to 24 hours.

2. What are the applications of TurboID in scientific research?

   TurboID finds extensive applications in molecular biology and biochemistry, particularly in the study of protein-protein interactions and the dynamics of protein networks within cells. It is especially valuable in identifying transient or weak protein interactions that are difficult to detect with traditional methods. Researchers use TurboID for various applications including mapping the components of specific cellular organelles, understanding signal transduction pathways, and exploring the changes in protein interactions under different physiological conditions or in disease states. Additionally, due to its effectiveness in living cells and organisms, TurboID is also applied in in vivo studies, providing insights into the biological processes at the organismal level.

3. What are the advantages of using TurboID over other proximity labeling methods?

   TurboID offers several advantages over traditional proximity labeling methods such as BioID or APEX. First, it requires much shorter incubation times to achieve effective labeling, ranging from 10 minutes to an hour, compared to the 24-hour incubation period often necessary for BioID. This rapid action reduces potential perturbations to cellular processes and minimizes the effects of overexpression toxicity. Secondly, TurboID demonstrates enhanced biotinylation efficiency, which improves the detection of low-abundance or transient protein interactions. Moreover, it functions efficiently at lower temperatures and in a broader range of biological systems, including less permeable cells and tissues, making it versatile for various experimental setups.

4. Are there any limitations or challenges associated with using TurboID?

   One primary challenge is the potential for background labeling, where non-specific biotinylation can occur due to the high activity of the enzyme. This can lead to false positives in protein interaction studies. Additionally, the high efficiency of TurboID might result in over-biotinylation, which could mask or alter the function of target proteins. Researchers must optimize experimental conditions carefully to balance labeling efficiency with specificity. Furthermore, like all biotin-based methods, TurboID requires the availability of free biotin in the cell, and its efficiency can be affected by biotin competition or depletion in the cellular environment.

5. Can TurboID be used in live animal models?

   Yes, TurboID can be effectively used in live animal models, expanding its utility beyond cell culture systems. This capability is particularly beneficial for in vivo studies where understanding protein interactions and dynamics within the actual physiological context is crucial. For example, researchers can express TurboID in specific tissues or cells of model organisms like mice through genetic engineering techniques. By administering biotin systemically or locally, proteins proximal to TurboID in these living organisms can be labeled and later analyzed to gain insights into tissue-specific or developmental processes.

6. What technical considerations should be taken into account when using TurboID?

   First, the concentration of biotin available in the cell or tissue environment needs to be carefully controlled, as insufficient biotin can lead to reduced labeling efficiency while excessive biotin might increase background noise. The expression level of TurboID also needs to be optimized to avoid toxicity and ensure it does not interfere with normal cellular processes. Additionally, the choice of the fusion strategy (whether TurboID is fused to a specific protein of interest or expressed alone) can affect the proximity labeling results. Proper controls should be established to differentiate between specific interactions and background labeling, such as using cells expressing TurboID without biotin or with a catalytically inactive version of the enzyme. Lastly, due to the rapid action of TurboID, timing the biotin administration correctly is crucial to capturing the dynamic changes in protein interactions, especially in studies involving transient cellular events or stimuli.

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