miniTurbo Interaction Analysis

What is miniTurbo?

miniTurbo is a powerful protein analysis tool that enables for the research of protein-protein interactions, protein-ligand interactions, and post-translational changes. This method combines TurboID principles with a smaller version of the standard TurboID approach. TurboID is a proximity labeling approach that identifies proteins that interact with a certain target protein in live cells. miniTurbo uses a similar method but adds numerous features that make it a useful tool for protein analysis. MiniTurbo improves efficiency, specificity, and flexibility over regular TurboID by incorporating unique adjustments.

E. coli biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red (Branon et al., 2018).

Principles and Workflow of miniTurbo

miniTurbo operates based on the principle of proximity labeling, where a target protein of interest is labeled in live cells with a biotin molecule. The labeled protein subsequently forms a complex with interacting proteins or ligands, which are then biotinylated. The biotinylated proteins can be easily purified and identified through affinity enrichment techniques, such as streptavidin-based pull-down assays.

Workflow of Our miniTurbo Service

Step 1: Design and Expression of miniTurbo Fusion Protein

The first step in the miniTurbo workflow is the design and expression of the miniTurbo fusion protein. The miniTurbo protein is genetically fused to the target protein of interest using molecular cloning techniques. This fusion construct ensures that miniTurbo is in close proximity to the target protein, enabling efficient labeling of interacting proteins.

Step 2: Expression of miniTurbo Fusion Protein in Living Cells

Once the miniTurbo fusion protein is constructed, it is expressed in the desired cellular system. This can be achieved through transient transfection or stable integration of the fusion construct into the genome of the host cells. Expression of the miniTurbo fusion protein allows for the formation of protein complexes and interactions within the cellular environment.

Step 3: Proximity Labeling with Biotin

The next step involves the proximity labeling process, where the miniTurbo fusion protein selectively biotinylates the proteins in close proximity. Biotin, a small molecule, is added to the cellular system as a substrate for the miniTurbo enzyme. Upon addition of biotin, the miniTurbo enzyme becomes activated and biotinylates neighboring proteins through a covalent bond.

The labeling reaction occurs in real-time, allowing for the capture of transient interactions that might be missed by other techniques. The biotinylation reaction is highly specific, with the biotin molecules being attached only to proteins that are physically close to the miniTurbo fusion protein.

Step 4: Affinity Enrichment

After the proximity labeling step, the biotinylated proteins need to be isolated and enriched for further analysis. Affinity enrichment techniques, such as streptavidin-based pull-down assays, are commonly employed for this purpose. Streptavidin, a protein with a high affinity for biotin, is conjugated to magnetic beads or other solid supports.

The biotinylated proteins are captured using streptavidin-coated magnetic beads, which selectively bind to the biotin molecules attached to the labeled proteins. Unbound proteins are removed through stringent washing steps, ensuring the isolation of specifically labeled proteins and minimizing background noise.

Step 5: Identification and Analysis

The final step in the miniTurbo workflow involves the identification and analysis of the enriched protein samples. The captured proteins are subjected to mass spectrometry or other analytical techniques, such as Western blotting or protein sequencing, to identify and quantify the interacting proteins.

Mass spectrometry-based proteomics is the most commonly used method for protein identification in miniTurbo analysis. The enriched proteins are digested into peptides, which are then analyzed by mass spectrometry. The resulting spectra are compared to protein databases, allowing for the identification of the interacting proteins.

Advanced bioinformatics tools are employed to analyze the mass spectrometry data and generate meaningful insights into protein functions, networks, and post-translational modifications. This analysis provides you with a comprehensive view of the protein interactions mediated by the target protein of interest.

Why Choose miniTurbo?

• Enhanced efficiency: miniTurbo exhibits improved labeling efficiency, enabling the detection of low-abundance interacting proteins that might be missed by other methods. This enhanced efficiency allows for a more comprehensive understanding of protein networks.
• Enhanced specificity: The modifications introduced in miniTurbo enhance its specificity, reducing non-specific labeling and background noise. This specificity ensures reliable and accurate identification of interacting proteins.
• Flexibility: miniTurbo can be applied to various cellular systems, including mammalian cells, yeast, and bacteria, making it adaptable to diverse research needs. The technology is compatible with a wide range of protein targets, facilitating the study of different protein classes.
• Minimal cellular perturbation: miniTurbo operates in live cells, minimizing any potential disruption to cellular processes. This feature enables the investigation of protein interactions and modifications within their native physiological contexts.
• Quantitative analysis: miniTurbo can be combined with quantitative proteomics techniques, such as stable isotope labeling or label-free quantification, allowing for the measurement of changes in protein interactions under different conditions.

Applications of miniTurbo Analysis

Protein-protein interaction studies: miniTurbo enables the identification and characterization of protein-protein interactions, shedding light on cellular pathways and protein complexes.

Protein-ligand interaction studies: miniTurbo can be employed to investigate protein-ligand interactions, facilitating the discovery and optimization of small-molecule drug candidates.

Mapping post-translational modifications: miniTurbo can identify and quantify post-translational modifications, providing valuable insights into the regulation of protein functions.

Exploration of protein networks: By uncovering interacting proteins and their networks, miniTurbo aids in understanding the global organization of proteins within cells, contributing to systems biology research.

Creative Proteomic offers comprehensive miniTurbo services to support you in their protein analysis endeavors. With years of expertise and cutting-edge technologies, Creative Proteomics ensures accurate and reliable results, helping you uncover the intricate world of proteins. Contact us to learn more.