Membrane proteins are one of the most important classes of proteins in biology, as they initiate complex signaling cascades and mediate the transport of various molecules across membranes, playing fundamental roles in both basic biology and clinical applications. A ubiquitin-based membrane yeast two-hybrid (MYTH) system has been developed for high-throughput analysis of protein-protein interactions involving membrane proteins.
Ubiquitin is a small polypeptide that acts as a tag to mark proteins for degradation and thus participates in the regulation of many processes in eukaryotic cells. Ubiquitin can be covalently attached to the N-terminus of a protein and subsequently recognized by ubiquitin-specific proteases (UBPs), leading to the proteolysis of the ubiquitin-conjugated protein. In the ubiquitin-based MYTH system, ubiquitin can be separated into two moieties: the N-terminal half (Nub) and the C-terminal half (Cub). The substitution of the isoleucine at position 3 of wild-type Nub with glycine (NubI mutated to NubG) greatly reduces its affinity for Cub. The Cub moiety is fused to an artificially designed LexA-VP16 transcriptional activator, forming a fusion protein Cub-LexA-VP16.
Due to the reduced affinity, NubG typically does not bind Cub, and the separated ubiquitin moieties cannot be recognized by UBPs, preventing the cleavage and release of the transcriptional activator. To detect protein-protein interactions, the target proteins are fused separately with NubG and Cub, generating the bait fusion protein (bait-Cub-LexA-VP16) and the prey fusion protein (prey-NubG). If the bait and prey interact, NubG and Cub are brought into proximity, leading to complexes and recognition by UBPs. The released LexA-VP16 then translocates into the nucleus and activates the transcription of a reporter gene.
Amerigo Scientific provides membrane yeast two-hybrid screening service for the membrane protein-protein interaction analysis using membrane protein cDNA libraries. Combing traditional yeast two-hybrid with next-generation sequencing technology, we offer a comprehensive solution to meet the need of analyzing interactions between membrane proteins and cytoplasmic proteins with high sensitivity and reliability.
Figure 1. Technique Principle of Our Membrane Two-hybrid Screening Service
Content of Our Y2H (Membrane System) Screening Service
The total workflow of our service typically requires about 60 working days.
| Content |
Timeline |
Core Deliverables |
Transmembrane Structure Analysis
- Comprehensive transmembrane structure analysis.
- Detailed planning.
|
1 Day |
- Positive clone sequences (≥15 sequences based on Sanger sequencing)
- Bait plasmid (optional)
- Comprehensive NGS data and bioinformatics analysis
- Detailed functional annotations (GO, KEGG) with interaction strength analysis
- Professional research report including:
Complete methodology;
Step-by-step experimental procedures;
High-resolution images;
Expert data interpretation |
Gene Synthesis (Codon Optimization) and Vector Construction
- Synthesize target genes with codon optimization.
- Clone genes into vectors.
|
15 Days |
Autoactivation Test of Bait Plasmid
- Perform self-activation testing to ensure no false positives.
|
10 Days |
Co-Transformation, Yeast Screening, and Sanger Sequencing
- Co-transform bait plasmid and library plasmid into yeast cells.
- Screen for positive interactions using selective media.
- Sequence 96 positive clones and analyze results.
|
20 Days |
Verification and Temporary Data Report
- Conduct rotation-based verification for result accuracy.
- Provide interim data report summarizing preliminary findings.
|
5 Days |
Next-Generation Sequencing and Final Report
- Perform NGS to analyze the complete interaction network.
- Generate and deliver a comprehensive project report, including all interaction data and bioinformatics analyses.
|
10 Days |
Key Advantages of Our Y2H (Membrane System) Screening Service
Our split-ubiquitin MYTH screening service has the following advantages:
- In vivo Interaction Detection: Protein interactions within cells are directly detected without the need for nuclear localization signals.
- Optimized Ubiquitin Domain: Small NubG and Cub moieties are utilized to minimize steric hindrance.
- Transcription-independent Activation: The use of UBPs to trigger LexA cleavage circumvents transcriptional activation limitations.
- Broad Applicability: The interactions of transmembrane, organelle, and cytoplasmic proteins can be analyzed.
- Maximum Sensitivity: The protocol is optimized to ensure minimal interference between interacting proteins.
- Comprehensive Analysis: Detailed functional annotations are provided by GO and KEGG frameworks.
- Fast Results: Our service requires short turnaround time and competitive price.
MYTH One-to-one Validation Service
Inquiry
In addition to a comprehensive MYTH screening service, Amerigo Scientific also offers a MYTH one-to-one interaction validation service for the precise analysis of specific protein-protein interactions within membrane systems. The total workflow of the one-to-one interaction validation service requires about 30 working days.
| Content |
Timeline |
Deliverables |
Transmembrane Analysis
- Transmembrane structure analysis.
|
1 Day |
- Recombinant plasmids (Optional).
- A digital experiment report.
|
Construction Phase
- Gene synthesis (codon optimization).
- Vector construction: Bait protein A vector constructed to pBT3-N/SUC/STE; protein B vector constructed to pPR3-N.
|
10 Days |
Transformation
- Recombinant plasmids transformed into yeast cells and autoactivation test performed.
|
7 Days |
Functional Validation
- Bait plasmid + pOst1-NubI used for validation.
|
3 Days |
Interaction Verification
- Set up positive and negative control groups for interaction verification.
|
10 Days |
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