TurboID Service

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 provide new TurboID services for protein-protein interaction exploration in vivo.

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What is TurboID?

TurboID is an advanced enzyme-based proximity labeling (PL) technique developed to identify protein-protein interactions (PPIs) with improved efficiency and speed. It builds upon the BioID method, which uses a promiscuous mutant of the E. coli biotin ligase (BirA) to biotinylate nearby proteins. TurboID introduces engineered biotin ligases optimized through yeast display, significantly enhancing the labeling process.

Fig. 1 Schematic representation of proximity-labeling systems.^1,5

Technical Workflow of TurboID

TurboID functions by tagging a protein of interest with a biotin ligase that catalyzes the attachment of biotin molecules to the lysine residues of neighboring proteins within approximately 10 nm. This biotinylation process creates a molecular "snapshot" of the protein's interaction environment. The biotinylated proteins are then purified using avidin-based affinity capture and identified through mass spectrometry (MS). Compared to traditional BioID, TurboID dramatically reduces the labeling time from 18-24 hours to as little as 10 minutes, making it more practical for dynamic or transient interaction studies.

Fig.2 Establishment of a TurboID-based proximity labeling system.^2,5

• TurboID Fusion Protein Construction: Fuse the target protein's gene with the TurboID enzyme gene and introduce it into cells.
• Protein Expression & Proximity Biotinylation: Express the TurboID fusion protein; biotinylate nearby proteins using biotin and ATP.
• Isolation of Biotinylated Proteins: Capture biotinylated proteins with streptavidin beads.
• Protein Identification & Analysis: Identify proteins via mass spectrometry and analyze protein-protein interaction networks.

Advantages of TurboID

The combination of speed, sensitivity, specificity, and adaptability makes TurboID a powerful tool for studying protein-protein interactions and exploring complex biological systems.

• Rapid labeling efficiency: TurboID significantly reduces labeling time compared to traditional BioID. While BioID requires 18-24 hours for effective biotinylation, TurboID completes the process in minutes (10-30 minutes).
• Versatility in All Biological Systems: TurboID can be applied to a wide range of biological systems including mammalian cells, yeast, parasites, plants, and living tissues, making it suitable for diverse areas of research.
• Non-Toxic and Physiologically Compatible: TurboID operates under non-toxic conditions, preserving cellular integrity during labeling.
• Improved detection of weak and transient interactions: TurboID's high catalytic efficiency enables it to capture weak and transient protein interactions that may be overlooked by other methods.
• Low background noise: TurboID produces low nonspecific signal, which improves the reliability and reproducibility of results.
• Compatible with mass spectrometry: Biotinylated proteins isolated by TurboID can be easily analyzed using mass spectrometry, providing a powerful platform for high-throughput protein identification.

Comparison of TurboID, Split-TurboID, BioID, and Its Variants

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 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.

Why Choose Creative Biolabs?

Creative Biolabs offers comprehensive TurboID-based Proximity Labeling Services, harnessing advanced biotinylation technology and extensive expertise in protein interaction mapping. TurboID's rapid and efficient labeling capabilities allow precise identification of protein-protein interactions, even in dynamic and transient systems. With cutting-edge tools and tailored strategies, we support research in therapeutic target discovery, biomarker identification, and pathway analysis, delivering high-quality results for complex interactome studies.

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Published Data

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

Fig. 3 TurboID-based identification of BIN2-proximal proteins in Arabidopsis.^3,5

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. 4 Establishment and optimization of TurboID labeling in the Chlamydomonas chloroplast using RBCS2-TurboID lines.^4,5

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.

Resources

Use the resources in our library to help you understand your options and make critical decisions for your study.

Avidity vs. Affinity: Understanding the Crucial Differences in Molecular Interactions

Enzyme-Linked Immunosorbent Assay (ELISA): A Versatile Tool for Biomedical Research and Diagnostics

Exploring the Intricate World of Chromatin: Structure, Function, and Its Roles in Diseases