TurboID Service

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Case Study

What Is TurboID

Proximity labeling, as a novel technology developed in recent years for studying protein-protein interactions and subcellular proteomes in live cells, has been successfully applied in various animal and plant systems. This approach involves fusing a bait protein with an enzyme possessing specific catalytic activity, which covalently attaches small molecule substrates (e.g., biotin) to endogenous proteins in proximity under the enzyme's catalysis. By enriching and analyzing the biotinylated proteins, the protein interactome with the bait can be obtained. TurboID, a biotin ligase generated through directed evolution, offers the advantages of being non-toxic to proteins and having high catalytic efficiency. Utilizing TurboID-mediated proximity labeling, the neighboring protein interactome of the protein of interest can be studied, revealing transient or weak protein-protein interaction networks within the cell and providing insights into complex biological processes.

Workflow of TurboID

1. Treatment of materials with biotin

2. Protein extraction and Western blotting to detect biotinylated proteins

Immunofluorescence labeling with streptavidin-fluorophore is used to detect biotinylated proteins before and after biotin treatment to assess cellular biotinylation.

3. Optimization of biotin treatment conditions

In the experimental process, selecting appropriate treatment conditions, including temperature, treatment time, and biotin concentration, is crucial for successfully identifying TurboID-bait's neighboring proteins.

4. Preparation of samples for proximity labeling protein mass spectrometry

Due to the strong affinity between biotin and streptavidin, streptavidin magnetic beads can be used to enrich biotinylated proteins, followed by mass spectrometry to identify the neighboring proteins biotin-labeled by TurboID.

Principle of TurboID Proximity Labeling

TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Through directed evolution optimization, TurboID exhibits higher activity (reducing the labeling time from 18 hours to 10 minutes) than previously described biotin ligase-based proximity labeling methods (such as BioID), enabling higher time resolution and broader in vivo applications. Proteins biotinylated by TurboID are then enriched using streptavidin beads and identified through mass spectrometry.

Principle of TurboID Proximity Labeling

Customer provides

Target cells and their culture protocol

Bait protein information

Deliverables

(1) Project completion report

(2) Protein elution silver-stained gel image

(3) Mass spectrometry raw data and analysis results

(4) Protein-protein interaction (PPI) network diagram

Efficient proximity labeling in living cells and organisms with TurboID

Journal: Nature Biotechnology

Published: 2018

Abstract:

Protein-protein interaction networks and subcellular localization of proteins are fundamental to all signal transduction and regulatory processes in cells. Proximity labeling (PL) by enzyme-catalyzed biotinylation has emerged as a novel approach to study the spatial and interaction features of proteins in live cells. However, current PL methods require over 18 hours of labeling time or the use of chemicals with limited cell permeability or high toxicity.

In this study, the authors used directed evolution in yeast to generate two hybrid mutants of the biotin ligase, TurboID, and miniTurbo, which exhibit higher catalytic efficiency than BioID or BioID2, and enable non-toxic and rapid biotinylation in cells, accomplished in just 10 minutes.

Results:

The authors characterized TurboID and miniTurbo in mammalian cells. When expressed in the cytosol of HEK 293T cells and labeled with exogenous biotin, affinity blotting demonstrated much faster endogenous biotinylation by TurboID and miniTurbo compared to BioID. The authors also compared the new hybrid ligase, BioID2, and found that the biotinylation products generated by TurboID within 10 minutes were almost equivalent to those produced by BioID/BioID2/BASU in 18 hours. TurboID and miniTurbo were also evaluated in comprehensive proteomic experiments, and the protein profiles derived within 10 minutes by TurboID and miniTurbo were similar in size and specificity to those obtained by BioID in 18 hours.

TurboID Service

Reference

Branon, T., Bosch, J., Sanchez, A. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36, 880–887 (2018).

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