Recombinant Bartonella quintana 30S ribosomal protein S10 (rpsJ)

How does the amino acid sequence of B. quintana rpsJ compare to other Bartonella species?

While the search results don't provide specific information about the B. quintana rpsJ sequence, we can infer from related research that ribosomal proteins are generally conserved across Bartonella species. For example, the 17-kDa protein from B. henselae shows antigenic cross-reactivity with B. quintana proteins, suggesting structural similarities across species . Research approaches for rpsJ would likely involve sequence alignment analysis across Bartonella species to identify conserved domains and species-specific variations that might be relevant for diagnostic applications or understanding species-specific functions.

What expression systems are most effective for producing recombinant B. quintana rpsJ?

Based on approaches used for other Bartonella proteins, several expression systems can be considered for rpsJ:

E. coli systems have been successfully employed for other Bartonella proteins, with yields of approximately 2.9 mg from 100 mL of bacterial culture reported for the B. henselae 17-kDa protein . Similar approaches using histidine-tagged constructs would likely be effective for rpsJ expression .

What is the optimal purification strategy for recombinant B. quintana rpsJ?

For optimal purification of recombinant B. quintana rpsJ, a strategy similar to that used for other Bartonella proteins would be recommended:

• Design an expression construct with an appropriate tag (histidine tag is commonly used)

• Express the protein in the selected system (E. coli being most cost-effective)

• Lyse cells under optimized conditions

• Purify using nickel-agarose column chromatography for His-tagged proteins

• Verify purity using SDS-PAGE (target >85% purity as achieved with other ribosomal proteins)

• Consider additional purification steps if needed (ion exchange, size exclusion)

The use of histidine tags has proven effective for the purification of other Bartonella proteins to near homogeneity, allowing for yields sufficient for downstream applications . For recombinant rpsJ, similar affinity chromatography approaches would likely achieve the necessary purity while maintaining protein structure and function.

How can researchers verify the structural integrity of recombinant rpsJ?

Verification of structural integrity for recombinant rpsJ should include:

• SDS-PAGE analysis to confirm molecular weight

• Western blotting with antibodies against the tag or the protein itself

• Circular dichroism (CD) spectroscopy to assess secondary structure

• Limited proteolysis to evaluate domain organization

• Functional assays to confirm biological activity (RNA binding assays)

• Mass spectrometry for precise mass determination and post-translational modification identification

For recombinant Bartonella proteins, maintaining antigenic integrity is crucial, as demonstrated with the B. henselae 17-kDa protein, which was recognized by sera from patients infected with both B. henselae and B. quintana . Similar approaches could validate the structural integrity of recombinant rpsJ.

How can recombinant rpsJ be employed in developing diagnostic assays for B. quintana infections?

Recombinant rpsJ could be utilized in diagnostic assays similar to other Bartonella proteins:

• Development of ELISA-based antibody detection systems using purified rpsJ as the capture antigen

• Western blot confirmatory tests for serodiagnosis

• Protein microarray systems for multiplex detection of antibodies against various Bartonella antigens

• Lateral flow assays for point-of-care diagnosis

The B. henselae 17-kDa protein has demonstrated effectiveness as an antigen for antibody-capture ELISA with 71.1% sensitivity and 93.0% specificity compared to immunofluorescent antibody assay . If rpsJ proves to be immunogenic and species-specific, it could potentially serve as a valuable diagnostic antigen, particularly in combination with other Bartonella antigens for improved sensitivity and specificity.

What approaches can be used to study rpsJ interactions with other ribosomal components?

To study rpsJ interactions with other ribosomal components, researchers can employ:

• Co-immunoprecipitation studies with tagged rpsJ to identify interacting partners

• Yeast two-hybrid or bacterial two-hybrid systems to screen for protein-protein interactions

• Surface plasmon resonance (SPR) to quantify binding kinetics

• Cryo-electron microscopy to visualize rpsJ position within the assembled ribosome

• Cross-linking mass spectrometry to map interaction interfaces

• Structural modeling based on homologous proteins from related species

Understanding these interactions could provide insights into B. quintana-specific translation mechanisms and potentially identify unique features that could be targeted for therapeutic development.

What are common challenges in expressing recombinant B. quintana ribosomal proteins?

Common challenges researchers face when expressing B. quintana ribosomal proteins include:

• Protein solubility issues (formation of inclusion bodies)

• Toxicity to expression host

• Codon usage bias affecting expression levels

• Proper folding challenges in heterologous systems

• Maintaining native conformational epitopes important for functional studies

For the biotinylated expression of proteins, researchers can utilize the AviTag-BirA technology, where BirA catalyzes the amide linkage between biotin and the specific lysine residue of the AviTag, as demonstrated for other recombinant Bartonella proteins . This approach may help maintain protein functionality while providing a useful tag for detection and purification.

How can researchers optimize the yield of soluble recombinant rpsJ?

To optimize soluble rpsJ yield, researchers should consider:

• Testing multiple expression hosts (E. coli BL21(DE3), Rosetta, Arctic Express)

• Evaluating different fusion tags (His, MBP, GST, SUMO)

• Optimizing expression conditions:

  • Induction at lower temperatures (16-25°C)

  • Reduced IPTG concentrations

  • Extended expression times

• Co-expression with chaperones to aid proper folding

• Testing various lysis and purification buffer compositions

The lyophilized powder format commonly used for recombinant proteins should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol recommended for stability, as suggested for other Bartonella recombinant proteins .

How does rpsJ function compare between different growth conditions of B. quintana?

B. quintana encounters varied environments during its infectious cycle, transitioning from the hemin-restricted human bloodstream to the hemin-rich body louse vector. Similar to how RpoE expression is regulated by temperature and hemin concentration , rpsJ expression and function may also be influenced by these environmental factors:

• Temperature shifts (37°C in human host vs. 28°C in body louse vector)

• Hemin concentration variations

• pH differences

• Nutrient availability changes

• Oxygen tension alterations

Research approaches would involve growing B. quintana under varying conditions, then analyzing rpsJ expression levels using RT-qPCR (similar to methods used for rpoE studies) and evaluating potential post-translational modifications or interaction partner changes .

What molecular techniques are most effective for studying rpsJ gene regulation?

For studying rpsJ gene regulation, researchers can utilize:

• Reporter gene assays (e.g., lacZ or GFP fusions) to monitor promoter activity

• Electrophoretic mobility shift assays (EMSA) to identify DNA-protein interactions

• Chromatin immunoprecipitation (ChIP) to identify regulatory proteins binding to the rpsJ promoter

• RNA-seq to measure transcriptional changes across different conditions

• CRISPR interference (CRISPRi) to modulate expression and study downstream effects

For quantitative analysis of gene expression, RT-qPCR has proven effective in B. quintana studies, as demonstrated in research on RpoE . This technique could be adapted for rpsJ expression studies using appropriate primers designed specifically for the rpsJ gene.

How might rpsJ be utilized in developing rapid detection methods for B. quintana?

Building on advances in B. quintana detection methods, rpsJ could potentially be incorporated into:

• Loop-mediated isothermal amplification (LAMP) assays targeting rpsJ gene sequences, which have shown high sensitivity and specificity for other B. quintana genes (as demonstrated with the groEL gene-based LAMP assay with a limit of detection of 125 fg/reaction)

• Protein-based lateral flow assays using anti-rpsJ antibodies

• Aptamer-based detection systems specific for rpsJ

• CRISPR-Cas biosensing platforms

The LAMP assay approach has demonstrated significantly higher sensitivity than qPCR for B. quintana detection in clinical samples (22.0% vs. 8.0% positivity rate in rhesus samples) , suggesting that nucleic acid-based detection methods targeting rpsJ could offer advantages for rapid, field-deployable testing.

What is the potential for rpsJ as a therapeutic target in B. quintana infections?

While not directly addressed in the search results, exploration of rpsJ as a therapeutic target could involve:

• Structural analysis to identify unique features compared to human ribosomal proteins

• Development of small molecule inhibitors that specifically disrupt rpsJ function

• Peptide-based approaches to interfere with rpsJ-RNA or rpsJ-protein interactions

• Antisense oligonucleotides targeting rpsJ mRNA

• Evaluation of combination approaches targeting multiple ribosomal proteins

Research in this area would require detailed structural characterization of rpsJ and validation of its essentiality for B. quintana survival and virulence.