This recombinant Rickettsia rickettsii 17 kDa surface antigen (omp) is produced in an E. coli expression system and includes the complete mature protein sequence from amino acids 20 to 159. Scientists have added two tags to make their work easier: a 10xHis tag at the N-terminus and a Myc tag at the C-terminus for detection and purification. SDS-PAGE analysis shows the protein achieves over 85% purity, which should work well for most research purposes.
The 17 kDa surface antigen—often called omp—appears to be quite important for how Rickettsia rickettsii functions. This protein seems central to how the bacterium sticks to and interacts with host cells during adhesion. Given its role in bacterial pathogenesis, it has become a popular target for researchers trying to understand rickettsial infections better and create new diagnostic approaches.
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.
Based on the provided information, the recombinant Rickettsia rickettsii 17 kDa surface antigen (omp) is expressed in E. coli, a prokaryotic system that is generally suitable for producing bacterial proteins like this surface antigen. As both Rickettsia and E. coli are prokaryotes, the protein has a reasonable probability of proper folding, but Rickettsia is an obligate intracellular bacterium with unique host adaptations, and its surface proteins may require specific folding environments or post-translational modifications that E. coli cannot provide. The protein is full-length mature (20-159aa) with dual tags (N-terminal 10xHis and C-terminal Myc) and >85% purity. However, the tags may interfere with native folding, particularly for a surface protein that might involve membrane association or protein-protein interactions. Since activity is unknown, the protein cannot be assumed to be correctly folded or bioactive without experimental validation of its antigenic properties or binding functionality.
1. Antibody Development and Validation Studies
The recombinant omp can serve as an effective immunogen for generating antibodies that recognize linear epitopes, even if the protein is misfolded. The dual tags facilitate purification and detection during antibody screening. However, if the protein is misfolded, antibodies may not recognize conformational or modification-dependent epitopes of native omp from R. rickettsii. Validation against native protein from Rickettsia cultures or infected cells is recommended to ensure physiological relevance. The 85% purity is sufficient for immunization protocols, but higher purity might be beneficial for some applications.
2. Protein-Protein Interaction Studies
The dual tags enable technical feasibility for pull-down assays, but this application requires caution. If the omp is misfolded (a possibility due to tag interference or E. coli's limitations for Rickettsia proteins), it may not interact physiologically with true binding partners (e.g., host cell receptors or other bacterial proteins). Identified interactions could be non-physiological artifacts. This application should only be pursued after confirming proper folding and activity through functional assays, such as binding studies with known partners.
3. ELISA-Based Binding Assays
This application is feasible for technical development but problematic for biological insights without folding validation. The tags allow for immobilization and detection in ELISA formats, but if omp is misfolded, binding data (e.g., ligand affinity or antibody specificity) may not reflect native behavior. This application should be limited to preliminary screening and require validation with properly folded omp or native protein for reliable biological conclusions.
4. Biochemical Characterization and Stability Studies
This application is well-suited and should be prioritized for initial assessment. Techniques like size-exclusion chromatography, circular dichroism spectroscopy, and thermal shift assays can directly evaluate the recombinant omp protein's folding state, oligomerization, and stability. These studies are valuable even if the protein is inactive, as they characterize the recombinant product itself and can inform about its suitability for other applications. The tags may influence results, but this can be accounted for in analysis.
Final Recommendation & Action Plan
Given the uncertainty in folding and bioactivity, recommend first performing biophysical and functional validation to assess the protein's quality. This should include: 1) Biophysical characterization (e.g., circular dichroism for secondary structure, size-exclusion chromatography for oligomeric state) to evaluate folding; 2) Functional validation using known binding partners or antigenicity assays (e.g., with antibodies against native omp) to confirm bioactivity. Antibody development can proceed immediately as the safest application. Avoid interaction and binding studies until proper folding is confirmed. For reliable functional data, consider using omp from eukaryotic expression systems or native sources if possible, as Rickettsia proteins may require host-specific factors. Always include appropriate controls, such as native omp or validated binding partners, to ensure physiological relevance.
