Recombinant Chlamydophila caviae 50S ribosomal protein L17 (rplQ)

What is the genomic context of the rplQ gene in Chlamydophila caviae?

The rplQ gene in C. caviae is part of the organism's 1,173,390 nucleotide genome, which has a GC content of 39.2% . The gene encodes the 50S ribosomal protein L17, one of the 1009 annotated genes in the C. caviae genome . As a ribosomal protein, rplQ would be among the conserved genes, likely part of the 798 genes that are conserved across all sequenced Chlamydiaceae family members . The genomic organization of C. caviae has been well characterized, with genes categorized into conserved hypothetical (31.7%) and hypothetical (8.3%) classifications .

To study the genomic context effectively, researchers should:

• Use comparative genomic approaches to identify conserved synteny with other Chlamydiaceae

• Analyze the promoter regions for regulatory elements using bioinformatic tools

• Consider the gene's location relative to the replication terminus, as genes in the replication termination region (RTR) often show greater variation across species

How is the structure of C. caviae L17 protein predicted to differ from other bacterial ribosomal L17 proteins?

The computed structure model of C. caviae 50S ribosomal protein L17 is available in the RCSB Protein Data Bank (identifier: AF_AFQ824N0F1) . This model suggests a typical ribosomal protein structure. When analyzing structural differences:

• Compare the structural features with other bacterial L17 proteins using structural alignment tools

• Focus on key functional domains and surface-exposed regions

• Analyze sequence conservation at the amino acid level, particularly in regions involved in RNA binding

Methodologically, researchers should employ multiple structure prediction algorithms, not relying solely on a single computational model. Experimental validation through techniques such as X-ray crystallography or cryo-EM would provide more definitive structural information.

What are the standard methods for expression and purification of recombinant C. caviae rplQ?

For recombinant expression of C. caviae rplQ:

• Design expression constructs with appropriate tags (His6, GST, or MBP) to facilitate purification

• Select a suitable expression system (commonly E. coli BL21(DE3) for ribosomal proteins)

• Optimize expression conditions:

  • Induction at OD600 0.6-0.8

  • IPTG concentration between 0.1-1.0 mM

  • Lower incubation temperatures (16-25°C) to improve solubility

• For purification, employ:

  • Affinity chromatography based on the fusion tag

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

Consider codon optimization for expression in heterologous systems, as C. caviae has a GC content of 39.2%, which differs from E. coli .

How might functional studies of C. caviae rplQ inform our understanding of its potential role in growth regulation?

Studies on ribosomal protein L17 in other organisms, particularly RpL17 in mouse vascular smooth muscle cells, have demonstrated significant growth inhibitory properties. RpL17 expression inversely correlates with cell proliferation, suggesting it functions as a growth inhibitor akin to a tumor suppressor . When designing experiments to investigate similar functions in C. caviae rplQ:

• Develop recombinant expression systems for C. caviae rplQ that allow controlled expression in mammalian cells

• Analyze cell cycle progression using flow cytometry to determine changes in G0/G1 and S phase populations

• Compare effects across different cell types to assess target cell specificity

• Employ siRNA knockdown approaches to test loss-of-function outcomes

Research on mouse RpL17 demonstrated that when expressed at higher levels (as in C3H/F mice compared to SJL mice), it correlated with slower cell growth . Similar functional studies with C. caviae rplQ could reveal whether this function is conserved across diverse species.

What strategies can be employed to study interactions between recombinant C. caviae L17 protein and other ribosomal components?

To investigate interactions between recombinant C. caviae L17 and other ribosomal components:

• Use pull-down assays with tagged recombinant L17 to identify binding partners

• Employ surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure binding affinities

• Conduct in vitro reconstitution experiments with purified components

• Apply chemical cross-linking followed by mass spectrometry to map interaction interfaces

For RNA interactions specifically:

• RNA immunoprecipitation (RIP) can identify RNA partners

• Electrophoretic mobility shift assays (EMSA) can confirm direct binding

• RNA footprinting can map the precise binding sites

Researchers should consider the complexities of ribosomal assembly when designing these experiments, as L17 interactions may be sequential and dependent on the presence of other factors.

What are the implications of comparative genomic analyses of rplQ across Chlamydiaceae for evolutionary studies?

Comparative genomic analyses of rplQ across Chlamydiaceae species can provide significant evolutionary insights:

To conduct meaningful evolutionary analyses:

• Perform BLAST score ratio (BSR) analyses as done for other C. caviae proteins

• Construct phylogenetic trees based on rplQ sequences

• Calculate selective pressure metrics (dN/dS) to identify conservation patterns

• Analyze synteny of the genomic regions containing rplQ

Similar to the analysis of guaBA-add genes which showed evidence of potential horizontal transfer between species , researchers should examine whether rplQ exhibits unusual phylogenetic relationships that might indicate evolutionary events like horizontal gene transfer.

What NIH guidelines must be followed when designing experiments with recombinant C. caviae rplQ?

When working with recombinant C. caviae rplQ, researchers must adhere to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules:

• The guidelines define recombinant nucleic acids as "molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a living cell"

• All recombinant DNA research within the United States falls under these guidelines

• Institutional Biosafety Committee (IBC) approval is required before initiating experiments

• Proper containment principles must be followed based on risk assessment

Methodologically, researchers should:

• Submit detailed experimental protocols to their IBC

• Identify appropriate biosafety levels for work with C. caviae components

• Document all safety measures and training procedures

• Maintain records of experimental approvals and modifications

The specific biosafety level may depend on whether you're working with the isolated gene, the recombinant protein, or intact organisms.

How can researchers effectively design expression vectors for optimal production of soluble recombinant C. caviae L17 protein?

To optimize expression vector design for soluble C. caviae L17 production:

• Analyze the protein's properties:

  • Molecular weight: Typically around 14-15 kDa for L17 proteins

  • Theoretical pI: Important for purification strategy

  • Hydrophobicity profile: Identifies potential solubility challenges

• Vector components to consider:

  • Promoter strength (T7, tac, or rhamnose-inducible for tight control)

  • Fusion tags (N-terminal versus C-terminal positioning)

  • Inclusion of solubility enhancers (SUMO, MBP, or TRX tags)

  • Codon optimization based on C. caviae's 39.2% GC content

• Expression strategies:

  • Test multiple constructs in parallel

  • Employ solubility screening approaches (like split-GFP systems)

  • Consider cell-free expression systems for difficult-to-express constructs

• Validation methods:

  • Western blotting to confirm expression

  • Activity assays to verify functional integrity

  • Circular dichroism to assess secondary structure

What analytical techniques are most appropriate for characterizing the functional properties of recombinant C. caviae L17?

For comprehensive characterization of recombinant C. caviae L17:

• Structural analysis:

  • Circular dichroism spectroscopy for secondary structure assessment

  • Thermal shift assays for stability determination

  • Limited proteolysis to identify domains and flexible regions

  • X-ray crystallography or cryo-EM for high-resolution structure

• Functional analysis:

  • RNA binding assays (filter binding, EMSA, fluorescence anisotropy)

  • In vitro translation assays to assess impact on protein synthesis

  • Cell proliferation assays based on findings from RpL17 studies

  • Protein-protein interaction studies using pull-downs or crosslinking

• Cellular localization:

  • Immunofluorescence microscopy

  • Subcellular fractionation

  • Proximity labeling approaches

Data from mouse RpL17 studies showed that expression inversely correlated with cells in S phase and increased cells in G0/G1 . Similar cell cycle analyses would be valuable for C. caviae L17 functional characterization.

How can researchers address solubility issues when expressing recombinant C. caviae L17 protein?

Solubility challenges are common with ribosomal proteins due to their native association with ribosomal RNA. To address solubility issues with C. caviae L17:

• Expression conditions modifications:

  • Reduce induction temperature to 16-20°C

  • Decrease inducer concentration

  • Use enriched media formulations (like Terrific Broth)

  • Co-express with chaperones (GroEL/ES, DnaK/J)

• Buffer optimization strategies:

  • Screen various pH conditions (typically 6.5-8.0)

  • Test different salt concentrations (150-500 mM NaCl)

  • Include stabilizing additives (glycerol, arginine, trehalose)

  • Add non-ionic detergents at low concentrations

• Refolding approaches:

  • Urea or guanidine denaturation followed by gradual dilution

  • On-column refolding during purification

  • Dialysis with decreasing denaturant concentration

• Alternative expression systems:

  • Insect cells for eukaryotic expression

  • Cell-free systems to avoid inclusion body formation

  • Specialized E. coli strains (SHuffle, Origami) for disulfide bond formation

What are the most effective methods for validating the biological activity of recombinant C. caviae L17 protein?

To validate biological activity of recombinant C. caviae L17:

• RNA binding validation:

  • Electrophoretic mobility shift assays with specific rRNA sequences

  • Filter binding assays to determine affinity constants

  • Competition assays with unlabeled RNA

• Ribosome incorporation:

  • In vitro reconstitution assays with bacterial ribosomal components

  • Complementation studies in L17-depleted bacterial systems

  • Sucrose gradient analysis to assess incorporation into ribosomal subunits

• Growth regulation activity:

  • Transfection into mammalian cells to assess proliferation effects

  • EdU incorporation assays to measure DNA synthesis

  • Cell cycle analysis by flow cytometry, looking for G0/G1 accumulation as observed with mouse RpL17

• Functional complementation:

  • Expression in L17-deficient bacterial strains to assess rescue of phenotypes

  • Competitive growth assays to measure fitness effects

Based on mouse studies, knockdown of RpL17 resulted in an 8-fold increase in proliferating cells in vivo , which could serve as a reference point for activity validation.

How can researchers distinguish between genuine biological effects and artifacts when studying recombinant C. caviae L17 in heterologous systems?

To distinguish genuine biological effects from artifacts:

• Include appropriate controls:

  • Empty vector controls

  • Unrelated proteins of similar size/properties

  • Inactive mutants of L17 (based on structure-function predictions)

  • Multiple cell lines or organisms to test conservation of effects

• Use multiple detection methods:

  • Combine biochemical, cellular, and in vivo approaches

  • Apply both gain-of-function and loss-of-function strategies

  • Use dose-response experiments to establish causality

  • Implement rescue experiments to confirm specificity

• Address potential confounding factors:

  • Evaluate tag interference by testing different fusion constructs

  • Monitor expression levels to avoid non-physiological overexpression

  • Assess cellular stress responses that might indirectly affect results

  • Consider off-target effects of genetic manipulation techniques

• Validate in native context:

  • Compare with native C. caviae protein behavior where possible

  • Correlate in vitro findings with observations in Chlamydia biology

How does the function of C. caviae L17 compare with ribosomal L17 proteins from other bacterial species?

A comparative functional analysis of L17 proteins should consider:

• Core ribosomal functions:

  • Contribution to ribosome assembly and stability

  • Involvement in tRNA positioning and peptidyl transferase activity

  • Interactions with other ribosomal components

• Extra-ribosomal functions:

  • Growth regulation properties (as observed with mouse RpL17)

  • Potential roles in stress response

  • Possible regulatory interactions with non-ribosomal partners

• Host-pathogen interactions:

  • Potential contributions to bacterial survival in host cells

  • Immunomodulatory properties

  • Roles in bacterial stress adaptation during infection

Research on mouse RpL17 demonstrated significant growth inhibitory properties, with expression inversely correlating with proliferation . Experiments should determine whether C. caviae L17 exhibits similar properties or has evolved distinct functions related to its obligate intracellular lifestyle.

What insights can be gained from studying the evolution of rplQ across the Chlamydiaceae family?

Evolutionary analysis of rplQ across Chlamydiaceae can reveal:

• Selection pressures:

  • Patterns of sequence conservation indicating functional constraints

  • Potential signatures of positive selection in specific lineages

  • Codon usage bias patterns that might reflect translational efficiency

• Phylogenetic relationships:

  • Congruence with species phylogeny versus evidence of horizontal gene transfer

  • Comparison with patterns observed in other ribosomal genes

  • Identification of clade-specific features related to lifestyle adaptations

• Structural evolution:

  • Conservation of key structural elements across the family

  • Lineage-specific structural adaptations

  • Co-evolution with interacting ribosomal components

The genome analysis of C. caviae revealed that while most genes show expected phylogenetic relationships, some gene clusters like guaBA-add showed evidence of horizontal transfer between rodent-associated Chlamydiae . Similar analysis of rplQ would determine whether it follows the expected evolutionary pattern or shows evidence of unusual evolutionary events.

How do differences in C. caviae L17 compared to human ribosomal L17 inform potential research applications?

Comparative analysis between C. caviae and human L17:

• Structural differences:

  • Identify unique surface features of bacterial L17

  • Map differences in RNA binding domains

  • Analyze potential epitopes for antibody development

• Functional distinctions:

  • Compare growth regulatory properties

  • Assess differences in interaction partners

  • Analyze differential responses to antibiotics or inhibitors

• Research applications:

  • Development of specific inhibitors targeting bacterial L17

  • Design of diagnostic tools based on unique features

  • Creation of recombinant systems to study evolutionary convergence/divergence

• Experimental approaches:

  • Recombinant expression of both proteins for side-by-side comparison

  • Chimeric protein construction to map functional domains

  • Cross-species complementation studies

Understanding these differences is crucial for applications seeking to exploit bacterial-specific features while avoiding cross-reactivity with human homologs.