Recombinant Escherichia coli O157:H7 Translocated intimin receptor Tir is expressed in E. coli, covering the extracellular domain region of 252-362 amino acids. The protein carries an N-terminal 6xHis-SUMO tag to help with purification and reaches purity levels above 90%, as confirmed through SDS-PAGE analysis. This product is made specifically for research purposes and is not intended for clinical applications.
The Translocated intimin receptor (Tir) appears to be a critical component in how Escherichia coli O157:H7 causes disease, particularly in its interactions with host cells. When Tir gets translocated into host cells, it seems to integrate into the host membrane, where it likely functions as a receptor for the bacterial adhesin intimin. This interaction may be essential for forming attaching and effacing lesions, which makes Tir an important target for researchers studying bacterial pathogenesis and how pathogens interact with their hosts.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Escherichia coli O157:H7 Translocated intimin receptor Tir is a bacterial virulence factor that requires precise folding, proper membrane association, and specific tertiary structure for its functional activity in host cell adhesion. The extracellular domain fragment (252-362aa) lacks the transmembrane and intracellular domains essential for full Tir functionality and proper structural context. The E. coli expression system is homologous to this bacterial protein, which increases the probability of correct folding. However, the large N-terminal 6xHis-SUMO tag (∼15 kDa) may sterically interfere with the protein's functional domains and intimin-binding interfaces. While the homologous expression system favors proper folding, the extracellular fragment nature and tag interference necessitate experimental validation of structural integrity and binding capability.
1. Antibody Development and Characterization Studies
This application is highly suitable as antibody development relies on antigenic sequence recognition rather than functional protein conformation. The extracellular domain provides specific epitopes for generating antibodies against the Tir intimin-binding region. The high purity (>90%) ensures minimal contamination-related issues during immunization protocols.
2. Protein-Protein Interaction Studies
This application carries a significant risk without proper folding validation. Tir's interaction with intimin requires the precise tertiary structure of the extracellular domain. If correctly folded (verified through intimin binding assays), the protein may identify physiological binding partners. If misfolded/unverified, there is a high risk of non-specific binding or failure to replicate genuine host-pathogen interactions. The SUMO tag may sterically block the intimin-binding site.
3. Structural and Biochemical Characterization
These studies are essential for determining folding status. Techniques should include circular dichroism spectroscopy to assess secondary structure, size-exclusion chromatography to evaluate oligomeric state, and thermal shift assays to determine stability. However, the SUMO tag may interfere with crystallization for high-resolution structural studies.
4. In Vitro Binding and Competition Assays
This application requires rigorous functional validation. Tir-intimin binding requires native conformation of the extracellular domain. If correctly folded and active (verified), limited binding studies may be possible. If misfolded/inactive (unverified), competition assays will yield biologically misleading results. The SUMO tag may physically prevent proper intimin access to the binding interface.
Final Recommendation & Action Plan
The homologous E. coli expression system provides favorable conditions for this bacterial virulence factor, but the extracellular domain fragment and large SUMO tag necessitate experimental validation before functional applications. Begin with Application 3 (Structural Characterization) to assess folding quality through CD spectroscopy, SEC, and validate intimin-binding capability using known interaction partners. Applications 2 and 4 require rigorous functional validation before proceeding. Application 1 (antibody development) can proceed immediately. Consider SUMO tag removal for critical binding studies to minimize steric interference. Always include appropriate controls and validate key findings with full-length Tir when possible for reliable host-pathogen interaction research.
