The study of protein-protein interactions is essential for deciphering cellular functions. Various methodologies exist for this purpose, ranging from traditional biochemical assays to advanced proteomic techniques. BioID and TurboID are proximity-dependent biotinylation methods that enable the identification of interacting proteins within a cellular context.
Select Services
BioID Technology
BioID technology is a novel biotin labeling approach that enables the identification of transient and low-affinity interactions. It provides a unique and powerful tool for studying protein-protein interactions, their dynamics, and the changes during disease progression.
Select Services
Workflow of BioID experiment with SCI1 as bait (Hollstein et al., 2022).
Principle of BioID
BioID technology, a powerful tool for investigating protein-protein interactions in living cells, operates on the principle of proximity-dependent biotinylation. The key components of BioID include a protein of interest fused to a promiscuous biotin ligase, BirA*, and exogenous biotin added to the cell culture medium. Upon expression in cells, the fusion protein facilitates the biotinylation of proximal proteins through BirA*-mediated enzymatic activity. This biotinylation occurs through the transfer of biotin molecules from free biotin to lysine residues within the vicinity of the bait protein. The biotinylated proteins are subsequently captured and isolated using streptavidin-based affinity purification methods, allowing for the identification and characterization of interacting partners.
Advantages and Limitations Analysis of BioID
- Sensitivity: BioID technology offers high sensitivity in detecting protein interactions, allowing for the identification of weak or transient associations that may be missed by other methods.
- Specificity: However, BioID may suffer from nonspecific biotinylation, wherein proteins in close proximity to the bait protein are erroneously labeled. This can introduce noise into the dataset and complicate data interpretation.
- Time: One drawback of BioID is the relatively long labeling times required to achieve sufficient biotinylation levels. Extended labeling periods may hinder the study of rapidly occurring protein interactions or dynamic cellular processes.
- Cost: BioID experiments can incur higher costs compared to some alternative methods due to the need for specialized reagents such as BirA* fusion constructs and biotin labeling reagents.
Application Examples of BioID
Protein-Protein Interaction Studies:
BioID technology has revolutionized the study of protein-protein interactions by enabling the mapping of interactomes within living cells. By fusing a protein of interest with BirA*, researchers can label and capture proteins that come into close proximity, providing valuable insights into the spatial and temporal organization of cellular signaling networks. For example, BioID has been utilized to elucidate the interactomes of key signaling pathways, such as those involved in cell proliferation, apoptosis, and differentiation. Additionally, BioID has facilitated the identification of dynamic protein complexes and regulatory networks implicated in various biological processes, including DNA replication, transcription, and vesicular trafficking. Through comprehensive interactome mapping, BioID aids in unraveling the complex web of protein interactions that underlie cellular function and dysfunction, offering novel targets for therapeutic intervention in diseases such as cancer, neurodegeneration, and infectious disorders.
Proteomics Research:
In the realm of proteomics, BioID serves as a powerful tool for profiling protein localization, dynamics, and function within cellular contexts. By selectively biotinylating proximal proteins, BioID enables the identification and characterization of protein complexes, organelle proteomes, and subcellular structures. For instance, BioID has been employed to delineate the proteome of specific cellular compartments, such as the nucleus, mitochondria, and endoplasmic reticulum, shedding light on their molecular composition and functional interactions. Moreover, BioID-based proximity labeling can capture dynamic changes in protein localization and interaction patterns in response to cellular stimuli or disease conditions. By coupling BioID with quantitative proteomic techniques, researchers can elucidate the dynamics of protein complexes and signaling pathways in a spatially and temporally resolved manner. This integrative approach holds promise for uncovering novel biomarkers and therapeutic targets for diseases characterized by aberrant protein-protein interactions, including metabolic disorders, autoimmune diseases, and cardiovascular conditions.
Select Services
TurboID Technology
Principle of TurboID
TurboID technology represents an innovative advancement in proximity-dependent biotinylation, aiming to overcome limitations associated with traditional BioID methodology. Central to TurboID is the optimization of the biotin ligase enzyme, BirA, to enhance its catalytic activity and substrate specificity. This improved enzymatic efficiency enables the rapid and efficient biotinylation of proximal proteins within living cells. Additionally, TurboID employs high-activity biotinylated substrates, which serve as efficient targets for BirA-mediated biotinylation. Following biotinylation, the labeled proteins are captured and isolated using streptavidin-based affinity purification methods, allowing for the identification and characterization of interacting partners.
Select Services
Schematic representation of proximity-labeling systems. (Yang, 2020)
Advantages and Limitations Analysis of TurboID
Improvements Over BioID:
TurboID offers several key improvements over BioID methodology. Firstly, the enhanced catalytic activity of BirA in TurboID results in significantly shorter labeling times compared to BioID, allowing for more rapid and time-sensitive experiments. This accelerated kinetics enable the capture of transient protein interactions and dynamic cellular processes with higher temporal resolution. Moreover, TurboID exhibits improved substrate specificity, reducing the likelihood of nonspecific biotinylation and minimizing background noise in the obtained datasets. These enhancements contribute to cleaner and more reliable results, enhancing the interpretability of TurboID-generated data.
Areas for Further Improvement:
Despite its advancements, TurboID technology continues to evolve, with ongoing efforts focused on further enhancing its performance and versatility. One area for improvement lies in optimizing the specificity of TurboID-mediated biotinylation, particularly in complex cellular environments with high background protein concentrations. Strategies to enhance substrate selectivity and minimize off-target labeling are actively pursued to improve the accuracy and reliability of TurboID-generated data. Additionally, refinement of labeling conditions and experimental protocols may be necessary to maximize the efficiency and consistency of TurboID-mediated biotinylation across different biological systems and experimental setups.
Application Examples of TurboID
In Vivo Protein-Protein Interaction Studies:
TurboID technology has emerged as a powerful tool for investigating protein-protein interactions within the complex milieu of living cells. By leveraging the enhanced catalytic activity of BirA* in TurboID, researchers can achieve rapid and efficient biotinylation of proximal proteins, allowing for the identification of dynamic interaction networks in vivo. For example, TurboID has been employed to elucidate the interactomes of key signaling pathways implicated in cell growth, differentiation, and disease progression. Through proximity labeling of protein complexes in live cells, TurboID enables the characterization of transient and weak interactions that may evade detection by traditional methods. Moreover, TurboID offers the advantage of accelerated labeling kinetics, making it particularly well-suited for studying rapid cellular processes and dynamic signaling events. By integrating TurboID with advanced imaging techniques such as fluorescence microscopy or mass spectrometry, researchers can gain insights into the spatial and temporal dynamics of protein interactions within living systems, shedding light on the underlying mechanisms of cellular function and dysfunction.
Proteomics Research:
TurboID technology holds significant promise for advancing proteomics research by facilitating the comprehensive profiling of protein-protein interactions and subcellular proteomes. Through targeted biotinylation of proximal proteins, TurboID enables the identification and characterization of protein complexes, organelle proteomes, and molecular pathways within their native cellular contexts. For instance, TurboID has been utilized to map the proteome of specific subcellular compartments, such as the plasma membrane, cytoskeleton, and nuclear envelope, providing valuable insights into their molecular composition and functional interactions. Additionally, TurboID-mediated proximity labeling can capture dynamic changes in protein localization and interaction patterns in response to cellular stimuli or disease states. By combining TurboID with quantitative proteomic techniques, researchers can unravel the complex networks of protein interactions underlying cellular processes and disease pathways. This integrative approach offers new opportunities for discovering biomarkers and therapeutic targets for a wide range of diseases, including cancer, neurodegenerative disorders, and infectious diseases.
Comparison of BioID and TurboID
Technical Principle Comparison:
BioID and TurboID share a common underlying principle of proximity-dependent biotinylation, wherein a bait protein fused to a biotin ligase catalyzes the biotinylation of neighboring proteins within living cells. Both techniques rely on the enzymatic activity of BirA* to transfer biotin moieties onto lysine residues of proximal proteins, allowing for their subsequent capture and identification. However, TurboID incorporates key improvements in the form of a mutated BirA* enzyme with enhanced catalytic activity, resulting in accelerated biotinylation kinetics compared to BioID. This heightened enzymatic efficiency enables TurboID to achieve rapid and efficient labeling of proximal proteins within minutes, offering advantages in terms of experimental speed and temporal resolution.
Performance Comparison:
- Sensitivity: Both BioID and TurboID exhibit high sensitivity in detecting protein-protein interactions, allowing for the identification of weak or transient associations. However, TurboID's enhanced catalytic activity confers a potential sensitivity advantage by facilitating more rapid and efficient biotinylation of proximal proteins.
- Specificity: BioID may suffer from nonspecific biotinylation, leading to the labeling of proteins that are not in direct proximity to the bait protein. In contrast, TurboID's improved catalytic efficiency may enhance specificity by reducing off-target labeling, resulting in cleaner datasets with reduced background noise.
- Time: TurboID offers significant advantages in terms of labeling time, with biotinylation occurring within minutes compared to the longer labeling periods required by BioID. This accelerated kinetics of TurboID can be particularly advantageous for studying dynamic cellular processes and transient protein interactions.
- Cost: The cost of experiments using BioID and TurboID may vary depending on factors such as reagent availability and experimental setup. While TurboID may require specialized reagents or constructs, its faster labeling kinetics may result in reduced overall experimental costs compared to BioID.
Scope of Applications Comparison:
BioID and TurboID each have distinct strengths and applications within the realm of protein-protein interaction analysis. BioID is well-suited for comprehensive mapping of protein interaction networks within living cells and has been widely employed in studies investigating complex signaling pathways, subcellular proteomes, and dynamic interaction dynamics. On the other hand, TurboID's rapid labeling kinetics make it particularly suitable for studying rapid cellular processes, transient protein interactions, and dynamic signaling events. Additionally, TurboID's enhanced specificity may be advantageous for studies requiring precise spatial and temporal resolution of protein interactions. Both techniques have found applications in various research fields, including cell biology, proteomics, and systems biology, contributing to our understanding of cellular function and disease mechanisms.
References
- Hollstein, Lucas S., et al. "Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora." Scientific Reports 12.1 (2022): 17727.
- Yang, X.X; et al. Proximity labeling: an emerging tool for probing in planta molecular interactions. Plant Communications. 2020, 2(2): 100137.
You may be interested in:
