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Cells Apr 2020BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the... (Comparative Study)
Comparative Study
BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.
Topics: A549 Cells; Animals; Biotinylation; Genetic Vectors; Humans; Ligases; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Proteomics
PubMed: 32344865
DOI: 10.3390/cells9051070 -
Nature Protocols Jan 2023Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait... (Review)
Review
Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or clustered regularly interspaced short palindromic repeats (CRISPR)-based tagging, thus prohibiting the use of such assays in cell types that are difficult to transfect or transduce. We recently developed an 'off-the-shelf' proximity biotinylation method that makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait-specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. In principle, any scientist can perform this protocol within 3 days, although generating the proteomics data requires access to a high-end liquid chromatography-mass spectrometry setup. Data analysis and data visualization are relatively straightforward and can be performed using any type of software that converts raw mass spectrometry spectra files into identified and quantified proteins. The protocol has been optimized for nuclear targets but may also be adapted to other subcellular regions of interest.
Topics: Biotin; Biotinylation; Streptavidin; Proteome; Ligases
PubMed: 36224470
DOI: 10.1038/s41596-022-00748-w -
Molecular Microbiology Mar 2022In the last 10 years, proximity-dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from... (Review)
Review
In the last 10 years, proximity-dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from specific protein complexes to entire compartments. This is achieved by using enzymes such as BirA* and APEX that are fused to proteins of interest and biotinylate proteins in their proximity. PDB techniques are now also increasingly used in apicomplexan parasites. In this review, we first give an overview of the main PDB approaches and how they compare with other techniques that address similar questions. PDB is particularly valuable to detect weak or transient protein associations under physiological conditions and to study cellular structures that are difficult to purify or have a poorly understood protein composition. We also highlight new developments such as novel smaller or faster-acting enzyme variants and conditional PDB approaches, providing improvements in both temporal and spatial resolution which may offer broader application possibilities useful in apicomplexan research. In the second part, we review work using PDB techniques in apicomplexan parasites and how this expanded our knowledge about these medically important parasites.
Topics: Biology; Biotinylation; Proteins
PubMed: 34587292
DOI: 10.1111/mmi.14815 -
Journal of Biochemistry Dec 2021Recent advances in biotinylation-based proximity labelling (PL) have opened up new avenues for mapping the protein composition of cellular compartments and protein... (Review)
Review
Recent advances in biotinylation-based proximity labelling (PL) have opened up new avenues for mapping the protein composition of cellular compartments and protein complexes in living cells at high spatiotemporal resolution. In particular, PL combined with mass spectrometry-based proteomics has been successfully applied to defining protein-protein interactions, protein-nucleic acid interactions, (membraneless) organelle proteomes and secretomes in various systems ranging from cultured cells to whole animals. In this review, we first summarize the basics and recent biological applications of PL proteomics and then highlight recent developments in enrichment techniques for biotinylated proteins and peptides, focusing on the advantages of PL and technical considerations.
Topics: Animals; Biotinylation; Humans; Mass Spectrometry; Organelles; Protein Binding; Protein Interaction Maps; Proteome; Proteomics; Secretome
PubMed: 34752609
DOI: 10.1093/jb/mvab123 -
Trends in Biochemical Sciences May 2017Protein biotinylation is a key post-translational modification found throughout the living world. The covalent attachment of a biotin cofactor onto specific metabolic... (Review)
Review
Protein biotinylation is a key post-translational modification found throughout the living world. The covalent attachment of a biotin cofactor onto specific metabolic enzymes is essential for their activity. This modification is distinctive, in that it is carried out by a single enzyme: biotin protein ligase (BPL), an enzyme that is able to biotinylate multiple target substrates without aberrant-off target biotinylation. BPL achieves this target selectivity by recognizing a sequence motif in the context of a highly conserved tertiary structure. One structural class of BPLs has developed an additional 'substrate verification' mechanism to further enable appropriate protein selection. This is crucial for the precise and selective biotinylation required for efficient biotin management, especially in organisms that are auxotrophic for biotin.
Topics: Biotin; Biotinylation; Humans; Ligases; Protein Processing, Post-Translational
PubMed: 28268045
DOI: 10.1016/j.tibs.2017.02.001 -
Methods in Molecular Biology (Clifton,... 1999
Review
Topics: Animals; Antibodies; Biotin; Biotinylation
PubMed: 10098164
DOI: 10.1385/1-59259-213-9:39 -
Molecular & Cellular Proteomics : MCP May 2020The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of... (Review)
Review
The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.
Topics: Animals; Biotinylation; Enzymes; Humans; Proteomics; Staining and Labeling; Substrate Specificity
PubMed: 32127388
DOI: 10.1074/mcp.R120.001941 -
Nature Protocols Dec 2020This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in...
This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase-related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein-protein interactions or organelle-organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5-7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).
Topics: Biotinylation; Mass Spectrometry; Protein Interaction Mapping; Staining and Labeling
PubMed: 33139955
DOI: 10.1038/s41596-020-0399-0 -
Methods in Enzymology 2014Biotin is a naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because biotin is small (244 Da), it can be conjugated to many...
Biotin is a naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because biotin is small (244 Da), it can be conjugated to many proteins without altering their biological activities. The biotinylated molecule can be detected in ELISA, dot blot, or Western blot methods (see Western Blotting using Chemiluminescent Substrates) using streptavidin or avidin probes.
Topics: Biotin; Biotinylation; Polyethylene Glycols; Proteins
PubMed: 24423271
DOI: 10.1016/B978-0-12-420070-8.00010-6 -
Methods in Molecular Biology (Clifton,... 2022Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein-protein interactions. They engage with specific ligands and... (Review)
Review
Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein-protein interactions. They engage with specific ligands and translocate to the nucleus, where they bind the DNA and activate extensive transcriptional programs. Therefore, gaining a comprehensive overview of the protein-protein interactions they establish requires methods that function effectively throughout the cell with fast dynamics and high reproducibility. Focusing on estrogen receptor alpha (ESR1), the founding member of the nuclear receptor family, this chapter describes a new lentiviral system that allows the expression of TurboID-hemagglutinin (HA)-2 × Strep tagged proteins in mammalian cells to perform fast proximity biotinylation assays. Key validation steps for these reagents and their use in interactome mapping experiments in two distinct breast cancer cell lines are described. Our protocol enabled the quantification of ESR1 interactome generated by cellular contexts that were hormone-sensitive or not.
Topics: Animals; Biotinylation; Hormones; Mammals; Protein Interaction Mapping; Receptors, Cytoplasmic and Nuclear; Reproducibility of Results
PubMed: 35612745
DOI: 10.1007/978-1-0716-2124-0_15