Creative Biolabs provides a novel technique, proximity-dependent biotin identification approach, recently described as BioID, for screening and identification of protein-protein interactions in vivo. It takes advantage of the ability of a mutant form of biotin ligase to promiscuously biotinylate proximal proteins, no matter directly interacting or vicinal ones.

The Brief Introduction of BioID

BioID, a unique and readily accessible method, allows detecting protein-protein interactions that occur in intact cells. Its mechanism is relying on a promiscuous biotin protein ligase, which is fused to a bait protein expressed in living cells and then biotinylated proximate endogenous proteins with excess biotin during a defined labeling period. This capability of biotinylation is irrespective of whether these interact directly or indirectly with the fusion protein of interest or merely located in the same subcellular neighborhood. Following biotin-affinity capture of streptavidin, biotinylated proteins can be selectively isolated and identified by mass spectrometry.

Fig.1 Diagram of basic BioID. (Varnaitė, R.; MacNeill, SA. 2016)

To date, BioID represents a promising new approach for kinds of cell types from diverse species not only in mammalian cells but also in cellular constituents, such as insoluble nuclear lamina and centrosome. It has become a powerful tool and notably applicable to study insoluble proteins, identify low affinity or transient interactions, and is amenable to temporal regulation.

The Background of BioID

The original guide of BioID is devised by a method named DamID, where the prokaryotic Dam methylase fused to the protein of interest monitors DNA-protein interaction in eukaryotes. Analogous to DamID, BioID protocols utilize BirA*, a mutant of prokaryotic Escherichia coli (E.coli) biotin ligase, to indiscriminately biotinylate proximal proteins. And BirA* is derived from a homologous enzyme BirA (R118G mutated), a 35-kD DNA-binding biotin protein ligase.

Biotinylation is a two-step reaction, where the biotin ligase makes use of biotin and ATP to produce a highly reactive biotinoyl-5′-AMP intermediate, which subsequently reacts with a specific lysine residue on the target protein, releasing AMP. In BioID, a wild-type biotin ligase BirA has stringent specificity for its substrate, but its adopted mutation, BirA*, affects biotin ligase activity (disordered loop) rendering it a promiscuous biotinylation enzyme and can covalently modify primary amines in the immediate vicinity of BirA*. For highly reactive and short-lived biotinoyl-5′-AMP, the zone of modification in BioID is thought to be only ~10 nm in labeling radius around the BirA*-tagged protein.

A Workflow of BioID

There are two main stages splitting out in a BioID process: (i) generate a BioID fusion protein vector for stable expression within a mammalian cell or desired cell line; (ii) utilize the stable cell line to proceed a large-scale BioID pull-down for validation of potential protein-interactors by mass spectrometry. The overall scheme of BioID steps in detail as follows:

Fig.2 Flowchart of the BioID. (Le Sage, V. et al. 2016)

a. Construct BioID expression vector ligating bait with BirA*
b. Stably express BioID fusion protein in cells
c. Induce biotinylation by incubating excess biotin
d. Perform cell lysis, protein denaturation under relatively harsh conditions
e. Isolate biotinylated proteins with streptavidin-sepharose by biotin-affinity capture
f. Identify candidate proteins using mass-spectrometry or immunoblot analysis

The Merits of BioID

It provides multiple advantages over traditional approaches for studying protein-protein interactions, including:

• BioID has been proved usefully to identify weak and transient interactions which occur in vivo that cannot be detected by Y2H or affinity purification.
• BioID can overcome solubility issues and is particularly suited to investigate insoluble or inaccessible protein structures, as compared to Y2H and complex purification.
• Biotin binds strongly to streptavidin permitting a washing condition with harsh detergents to eliminate mostly nonspecific bindings.
• Non-interacting but neighboring proteins are also labeled, which gives valuable information on the proteomic landscape surrounding the target protein in native environments.
• Because of BioID fusion proteins expressed theoretically in any cells, there’s less false positive or negative displayed for detection with more relevant context.
• The mechanism of BioID action is well suitable for the study of cellular processes that are dynamic in space and time, as well as providing kinetic data.

Significantly, it is worthy to note that BioID biotinylation is a mark of potential proximity and not an evidence for physical interactions. Thus, Creative Biolabs could also provide subsequent tests, like immunoprecipitation assays, to further validate proximity interactors identified by BioID and determine which reflect direct interactions in the physiological condition of cells.

Other optional Protein-Protein Interaction (PPI) Assay services:

• Two Hybrid Services
• Protein-Fragment Complementation Assay (PCA) 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
• TurboID
• Y2H-Based Drug-Target Interaction Identification
• Yeast Three-Hybrid (Y3H) for Protein Interaction Identification

Published Data

• Identification of LSD1/CoREST Associated Proteins Using BioID

Fig. 3 CoREST complex members tagged with BirA* expressed in HEK293T cells biotinylated proximal proteins. (Claire E. Barnes, 2022)

Lysine specific demethylase 1 (LSD1), as a part of the CoREST complex, regulates gene expression together with the REST co-repressor (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic sites through a large number of transient interactions between core components, chromatin-related factors, and transcription factors. In this paper, using three different cell types, the researchers used proximity-dependent biotin identification (BioID) for four different members of the CoREST complex to recognize an integrated network of LSD1/CoREST-related proteins. In HEK293T cells, researchers identified 302 CoREST-related proteins. At the same time, they replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early and late differentiation environments. This examines the dynamic properties of the CoREST interaction group in the primary cell type. Researchers also identified 156 LSD1-related proteins, 67 of which were constitutively associated at all three time points, meaning that most of the interacting proteins were dynamic and cell type dependent. Overall, researchers conducted 16 independent BioID assays on LSD1 using three different types of cells, producing a clear network of proteins associated with LSD1.

• Identification of Phytaspase Interactors via the BioID Approach

Fig. 4 Nicotiana tabacum phytaspase (NtPhyt)-TurboID and signal peptide (SP)-TurboID proteins utilized for the identification of phytaspase interactors. (Anastasia D. Teplova, 2021)

Because of their digestive activity and regulatory function, proteolytic enzymes play an important role in all aspects of plant development, including senescence. Here, researchers used a proximity-dependent biotin identification (BioID) assay to identify the protein chaperones of phytic acid proteases in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase, TurboID. And several candidate protein interactions of phytic acid protease have been identified. These are mainly soluble residues of endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticulin-3, its gene is characterized by increased expression in senescent leaves, and its direct interaction with phytic acid protease was confirmed in the in vitro binding test using purified protein. In addition, a significant change in the post-translational modification of calreticulin-3 was observed in plant cells overproduced by phytic acid protease.

FAQ

1. What is proximity-dependent biotin identification (BioID) and how does it work?

   BioID is used in molecular biology to study protein interactions within living cells. It involves the use of a promiscuous biotin ligase that is fused to a protein of interest. This biotin ligase, when expressed in cells, catalyzes the biotinylation of nearby proteins, effectively labeling them. The biotinylated proteins can then be isolated and identified using streptavidin-based methods. This approach allows researchers to capture and identify proteins that are in close proximity to the protein of interest, providing insight into protein interaction networks and cellular organization.

2. What are the advantages of using BioID over other protein-protein interaction methods?

   Firstly, it does not require direct physical interactions between proteins, as it can identify proteins within a proximity of approximately 10 nanometers. This is particularly useful for detecting transient or weak interactions that are often missed by other methods. Additionally, BioID can be conducted under physiological conditions within living cells, providing a more accurate representation of protein interactions and dynamics in their native cellular context. Lastly, BioID is relatively straightforward and can be applied to a wide range of biological systems, making it a versatile tool for protein interaction studies.

3. What types of biological questions can BioID help answer?

   BioID is particularly useful for exploring the molecular architecture of cells, understanding protein networks, and investigating cellular processes. It can help answer questions about the spatial organization of proteins within specific cellular compartments, identify novel components of protein complexes, or reveal proteins associated with particular cellular functions such as signaling pathways, organelle biogenesis, or disease mechanisms. By revealing proximity-based interactions, BioID can also assist in identifying potential targets for therapeutic intervention and provide insights into the molecular basis of diseases.

4. How is BioID adapted for different experimental needs?

   BioID has been adapted in various forms to enhance its versatility and specificity. For instance, a more rapid version called TurboID has been developed, which offers faster biotinylation speeds, allowing for shorter labeling times and reducing potential artifacts from overexpression and prolonged biotin ligase activity. Additionally, miniTurbo, a smaller version of TurboID, has been created to minimize the effects of the tag size on the function of the protein of interest. These adaptations make BioID suitable for dynamic studies where temporal resolution is crucial, such as monitoring changes in protein interactions during cell signaling events or other rapid cellular processes.

5. Can BioID be used in tissues or only in cell cultures?

   Originally developed for use in cell culture systems, BioID has been successfully adapted for use in tissue samples as well. This adaptation allows researchers to study protein interactions within more complex biological contexts, including multicellular environments and different organ systems. The application of BioID in tissues can provide insights into the spatial and temporal dynamics of protein networks in vivo, which is crucial for understanding physiological processes and disease states. However, applying BioID to tissues involves additional challenges, such as ensuring efficient delivery and expression of the biotin ligase fusion protein, and requires careful experimental design to obtain meaningful results.

6. What are the implications of using BioID for drug discovery and development?

   BioID can significantly impact drug discovery by identifying novel targets and elucidating protein interaction networks that are critical for disease mechanisms. By mapping these interactions in disease states versus healthy states, researchers can identify key proteins that may serve as potential drug targets. Furthermore, BioID's ability to capture transient and weak interactions provides a more comprehensive understanding of the protein complexes involved in signaling pathways and disease progression, offering insights into the molecular underpinnings that can be leveraged for therapeutic intervention. This makes BioID an invaluable tool in the early stages of drug development, especially for diseases with complex pathologies such as cancer and neurodegenerative disorders.

7. How does the spatial resolution of BioID compare to other proximity labeling methods?

   BioID generally has a spatial resolution of about 10 nanometers, which is sufficient to identify proteins within the same protein complex or organelle. However, compared to other proximity labeling methods, BioID offers slightly better spatial precision. This finer resolution is advantageous for dissecting dense protein networks and can be critical in densely packed cellular environments, like synaptic junctions or the nuclear periphery.

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