Recombinant Chlamydophila caviae 50S ribosomal protein L16 (rplP)

What is rplP and what functional role does it play in Chlamydophila caviae?

rplP is a 50S ribosomal protein L16 that belongs to the universal ribosomal protein uL16 family. It plays a critical role in the ribosomal complex by binding to 23S rRNA and making contacts with the A and possibly P site tRNAs . As part of the large ribosomal subunit, rplP contributes to the structural integrity of the ribosome and participates in the process of protein synthesis, which is essential for bacterial survival and replication. In Chlamydophila caviae, this protein is particularly important given the organism's complex biphasic developmental cycle, which requires extensive regulation of protein synthesis during transitions between elementary body (EB) and reticulate body (RB) forms .

How conserved is rplP across Chlamydia species?

Based on phosphoproteomic analysis, 41 of 42 C. caviae phosphoproteins were found to be present across various Chlamydia species . This high degree of conservation suggests that rplP likely maintains similar structural and functional characteristics throughout the Chlamydia genus. The conservation reflects the essential nature of ribosomal proteins in bacterial physiology and indicates that research findings on C. caviae rplP may have broader implications for understanding other Chlamydia species, including human pathogens like C. trachomatis and C. pneumoniae .

What are the key biochemical characteristics of recombinant C. caviae rplP?

Recombinant C. caviae rplP has the following key characteristics:

• Full-length protein consists of 138 amino acids

• Molecular weight of approximately 15.8 kDa

• Complete amino acid sequence: MLMPKRTKFRKQQKGQFAGLSKGATFVDFGEFGMQTLERGWVTSRQIEACRVAINRYLKRKGKVWIRVFPDKSVTKKPAETRMGKGKGAPDHWVAVVRPGRILFEVANVSREDAQDALRRAAAKLGIRTRFVKRVERV

• Belongs to the universal ribosomal protein uL16 family

• Functions by binding to 23S rRNA and interacting with tRNA at the A and P sites

What post-translational modifications have been identified in C. caviae rplP?

While specific post-translational modifications of rplP were not directly identified in the search results, the phosphoproteomic analysis of C. caviae revealed numerous phosphorylated proteins in both elementary bodies (EB) and reticulate bodies (RB) . Phosphorylation appears to be a significant post-translational modification in Chlamydia species, potentially regulating protein function during different developmental stages. The study identified 42 non-redundant phosphorylated proteins, with 34 in EBs and 11 in RBs, suggesting stage-specific regulation . Researchers investigating rplP should consider potential phosphorylation sites that might affect its function or interactions within the ribosomal complex.

What expression systems are most effective for producing recombinant C. caviae rplP?

Based on the search results, recombinant C. caviae rplP has been successfully expressed in both yeast and E. coli expression systems . When designing an expression protocol, researchers should consider:

• Yeast expression systems: Used for the commercial product described in search result , these systems can provide eukaryotic post-translational modifications, though these may differ from bacterial modifications.

• E. coli expression systems: Commonly used for bacterial proteins, E. coli systems typically offer high yield and simplicity. The search result mentions E. coli synthesis of rplP.

The choice between these systems should be guided by the specific research requirements, including the need for post-translational modifications, protein folding considerations, and downstream applications.

What are the recommended methods for purification and quality assessment of recombinant rplP?

For optimal purification and quality assessment of recombinant rplP, researchers should consider:

• Purification methods:

  • Affinity chromatography using appropriate tags (note that "tag type will be determined during the manufacturing process" )

  • Size exclusion chromatography to ensure homogeneity

• Quality assessment:

  • SDS-PAGE to confirm protein size (expected at approximately 15.8 kDa) and purity >85%

  • Western blot analysis for specific detection

  • Mass spectrometry to confirm protein identity

It's important to note that the final purified product should demonstrate >85% purity by SDS-PAGE according to commercial standards .

What are the optimal storage conditions for maintaining rplP stability?

For optimal stability of recombinant rplP, the following storage recommendations should be followed:

• Short-term storage (up to one week): Store working aliquots at 4°C

• Long-term storage: Store at -20°C/-80°C with glycerol as a cryoprotectant

  • Recommended glycerol concentration: 5-50% (default final concentration: 50%)

• Reconstitution guidelines:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to the recommended final concentration

  • Aliquot for long-term storage

The shelf life for liquid form is approximately 6 months at -20°C/-80°C, while lyophilized form can maintain stability for up to 12 months at -20°C/-80°C .

How can researchers address issues of protein degradation during experimental procedures?

To minimize degradation during experimental procedures, researchers should:

• Avoid repeated freeze-thaw cycles, as this is not recommended for maintaining protein integrity

• Work with aliquoted samples rather than the stock solution

• Keep the protein on ice during experiments

• Consider adding protease inhibitors to prevent degradation by contaminant proteases

• Use freshly prepared buffers and reagents

• Monitor protein stability using analytical techniques such as SDS-PAGE before critical experiments

How can recombinant rplP be used to study the developmental cycle of Chlamydia?

Recombinant rplP can serve as a valuable tool for investigating the Chlamydia developmental cycle through several approaches:

• Developmental stage-specific protein expression:

  • Comparing rplP expression levels between elementary bodies (EB) and reticulate bodies (RB) using quantitative proteomics

  • Investigating rplP interactions with other ribosomal components during developmental transitions

• Post-translational modifications:

  • The phosphoproteomic analysis revealed stage-specific protein phosphorylation patterns in C. caviae

  • Researchers can investigate whether rplP undergoes differential phosphorylation during the developmental cycle

  • Analysis of how potential modifications might affect ribosomal function during different stages

• Ribosomal assembly studies:

  • Using recombinant rplP to study ribosome assembly mechanisms specific to Chlamydia

  • Investigating differences in translation efficiency between developmental stages

What are the key considerations when using C. caviae as a model organism for chlamydial infection studies?

When using C. caviae as a model organism for chlamydial infection studies, researchers should consider:

• Biological relevance:

  • C. caviae was first isolated from the conjunctiva of laboratory guinea pigs and serves as a model for chlamydial ocular infection

  • It produces a trachoma-like disease in guinea pigs following repeated infection

• Experimental advantages:

  • Allows quantification of gross pathological responses in the conjunctiva over the course of infection

  • Pathological responses can be correlated with organism isolation from ocular swabs

  • Abundant conjunctival tissue is available for histopathologic, flow cytometric, and gene expression studies

• Immune response considerations:

  • C. caviae infection in guinea pigs shows neutrophil involvement in the pathological response

  • Neutrophil depletion studies have shown altered adaptive immune responses and decreased ocular pathology

• Cross-species comparisons:

  • High conservation of proteins (including ribosomal proteins) across Chlamydia species suggests findings may be applicable to other species

What methods are most effective for studying rplP interactions with other ribosomal components?

To effectively study rplP interactions with other ribosomal components, researchers should consider:

• Structural biology approaches:

  • X-ray crystallography to determine the three-dimensional structure of rplP alone or within the ribosomal complex

  • Cryo-electron microscopy (cryo-EM) for visualization of intact ribosomal complexes

  • Nuclear magnetic resonance (NMR) spectroscopy for dynamic interaction studies

• Biochemical interaction methods:

  • RNA binding assays to investigate rplP interaction with 23S rRNA

  • Co-immunoprecipitation to identify protein-protein interactions within the ribosomal complex

  • Cross-linking studies to capture transient interactions

• Functional assays:

  • In vitro translation assays to assess the impact of rplP on protein synthesis

  • Mutagenesis studies to identify critical residues for rplP function

  • Ribosome profiling to analyze translation dynamics

How can phosphoproteomic analysis be applied to study post-translational modifications of rplP?

Phosphoproteomic analysis can be applied to study potential post-translational modifications of rplP using the following methodologies:

• Sample preparation techniques:

  • 2D gel electrophoresis coupled with phosphoprotein staining (as used in the C. caviae phosphoproteome study)

  • Phosphopeptide enrichment using techniques such as immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO₂) chromatography

• Analytical approaches:

  • MALDI-TOF/TOF analysis for identification of phosphorylated proteins

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for high-sensitivity phosphopeptide detection

  • Multiple reaction monitoring (MRM) for targeted quantification of specific phosphorylation sites

• Comparative analysis between developmental stages:

  • Differential phosphorylation patterns between elementary bodies (EB) and reticulate bodies (RB)

  • Investigation of kinases responsible for phosphorylation

  • Functional implications of phosphorylation on protein activity

How can researchers address challenges in distinguishing between host and bacterial proteins in Chlamydia studies?

Distinguishing between host and bacterial proteins represents a common challenge in Chlamydia studies. Researchers can address this issue through:

• Purification strategies:

  • Implement more rigorous purification protocols to minimize host cell contamination

  • Consider differential centrifugation techniques to separate bacterial cells from host components

• Analytical approaches:

  • Use bioinformatic tools to distinguish bacterial from eukaryotic proteins based on sequence analysis

  • Apply targeted proteomics approaches focusing specifically on bacterial proteins

  • Consider stable isotope labeling to differentiate host and bacterial proteins

• Common contaminants to monitor:

  • Mouse proteins were identified in both EB and RB preparations in the phosphoproteomic study

  • Many of the mouse protein contaminants detected are known to be phosphorylated

  • Host cell protein contamination is recognized as a common problem in Chlamydia proteome studies

What are the main technical challenges in working with recombinant Chlamydial proteins and how can they be overcome?

The main technical challenges in working with recombinant Chlamydial proteins include:

• Protein solubility and folding:

  • Challenge: Bacterial proteins may form inclusion bodies or misfold in heterologous expression systems

  • Solution: Optimize expression conditions (temperature, inducer concentration), use solubility tags, or consider refolding protocols

• Post-translational modifications:

  • Challenge: Recombinant expression systems may not reproduce native bacterial modifications

  • Solution: Select appropriate expression systems or consider in vitro modification approaches

• Protein yield:

  • Challenge: Some Chlamydial proteins express poorly in standard systems

  • Solution: Codon optimization for the expression host, use of specialized expression strains, or optimization of culture conditions

• Functional activity:

  • Challenge: Ensuring the recombinant protein maintains native activity

  • Solution: Develop appropriate functional assays to validate protein activity post-purification