Recombinant Chlamydophila caviae UPF0301 protein CCA_00630 (CCA_00630)

What is Recombinant Chlamydophila caviae UPF0301 protein CCA_00630?

Recombinant Chlamydophila caviae UPF0301 protein CCA_00630 is a full-length protein (189 amino acids) originally identified in Chlamydophila caviae strain GPIC and produced in recombinant expression systems for research purposes . This protein belongs to the UPF0301 family, designated with the UniProt accession number Q822P9 . The term "UPF" (Uncharacterized Protein Family) indicates that while the protein has been identified and sequenced, its specific biological function remains not fully characterized. The recombinant version is typically expressed in systems such as yeast or E. coli to generate sufficient quantities for research applications .

What expression systems are recommended for producing CCA_00630?

Multiple expression systems can be utilized for producing recombinant CCA_00630, each with distinct advantages depending on research objectives. E. coli and yeast expression systems typically offer the highest yields and shortest production timeframes, making them cost-effective choices for many applications . For research requiring proper post-translational modifications that may be critical for protein function or structural studies, insect cell expression with baculovirus vectors or mammalian cell expression systems are recommended alternatives . The choice of expression system should be guided by the specific research questions being addressed, particularly whether native folding and post-translational modifications are essential for the planned experiments.

How should reconstituted CCA_00630 be stored to maintain stability?

For optimal stability of recombinant CCA_00630, several evidence-based storage protocols should be followed. After reconstitution, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and aliquot the protein solution for long-term storage at -20°C to -80°C . This approach minimizes damage from freeze-thaw cycles. For working stocks needed within a week, storing aliquots at 4°C is acceptable . The shelf life varies by preparation method: liquid preparations typically remain stable for 6 months when stored at -20°C to -80°C, while lyophilized preparations maintain stability for approximately 12 months under the same storage conditions .

What factors affect the stability of recombinant CCA_00630?

Multiple factors influence the stability of recombinant CCA_00630, including storage temperature, buffer composition, protein concentration, and freeze-thaw cycles. Lower temperatures (-80°C) generally provide better long-term stability than higher temperatures . The buffer composition significantly impacts stability, with glycerol functioning as a cryoprotectant that helps maintain protein structure during freezing . Repeated freeze-thaw cycles should be strictly avoided as they promote protein denaturation and aggregation . For research requiring multiple uses of the same protein preparation, creating single-use aliquots during initial reconstitution is strongly recommended to preserve protein integrity throughout the research timeline.

How does CCA_00630 relate to the broader context of variable proteins in Chlamydophila species?

CCA_00630 should be examined within the context of protein variability in Chlamydophila species, which exhibit significant genomic plasticity due to recombination hotspots . Chlamydiaceae are characterized by their diverse vertebrate host range, tissue tropism, and disease manifestations . Research has identified that Chlamydophila pneumoniae genomes contain a greater potential for recombination compared to Chlamydia trachomatis or Chlamydia muridarum . While CCA_00630 is not specifically identified as part of the highly polymorphic ppp family in C. pneumoniae, understanding the patterns of genetic variation in Chlamydophila species provides important context for studying this protein's evolutionary conservation and potential functional significance across species barriers .

What methodological approaches are recommended for functional characterization of CCA_00630?

A comprehensive functional characterization of CCA_00630 requires multiple complementary approaches:

• Comparative genomics analysis: Align CCA_00630 sequences across multiple Chlamydia species to identify conserved domains and predict functional relevance based on evolutionary conservation .

• Structural biology: Determine the three-dimensional structure through X-ray crystallography, cryo-EM, or NMR spectroscopy to identify potential functional domains and binding sites.

• Protein interaction studies: Perform pull-down assays, yeast two-hybrid screening, or co-immunoprecipitation to identify binding partners that might suggest functional pathways.

• Gene knockout or knockdown: Generate mutant Chlamydia strains with altered CCA_00630 expression to observe phenotypic effects on bacterial growth, infection, or pathogenesis.

• Immunolocalization: Use antibodies against CCA_00630 to determine its subcellular localization during different stages of the chlamydial developmental cycle.

These methodological approaches, used in combination, can provide insights into the biological role of this currently uncharacterized protein.

What is known about potential post-translational modifications of CCA_00630?

While specific post-translational modifications (PTMs) of CCA_00630 have not been definitively characterized in the provided search results, research approaches should consider the potential importance of PTMs in this protein's function. Expression in eukaryotic systems such as insect cells with baculovirus or mammalian cells can facilitate many of the post-translational modifications necessary for correct protein folding and activity retention . Common PTMs to investigate in Chlamydial proteins include phosphorylation, which can regulate protein activity, and glycosylation, which may be relevant for host-pathogen interactions. Methodologically, mass spectrometry-based proteomics approaches would be the gold standard for comprehensive PTM mapping, potentially revealing modifications that influence CCA_00630's stability, localization, or functional interactions within the bacterial cell or during host infection.

How can researchers optimize the purification protocol for CCA_00630 to obtain higher purity than the standard >85% SDS-PAGE purity?

Researchers seeking higher purity levels of CCA_00630 beyond the standard >85% SDS-PAGE purity should implement a multi-step purification strategy:

• Initial affinity chromatography: Leverage the recombinant tag (determined during the manufacturing process) for primary purification.

• Secondary ion-exchange chromatography: Separate proteins based on charge differences to remove contaminants with similar affinity but different charge properties.

• Size exclusion chromatography: Further purify based on molecular size to remove aggregates and lower molecular weight contaminants.

• Optimized buffer conditions: Adjust salt concentration, pH, and additives to enhance stability and solubility during purification.

• Quality control: Implement rigorous purity assessment using multiple methods beyond SDS-PAGE, such as analytical size exclusion chromatography, dynamic light scattering, and mass spectrometry.

This methodological approach can yield preparations exceeding 95% purity, which is critical for structural studies and certain functional assays where contaminants could confound results.

What approaches should be used to investigate potential interactions between CCA_00630 and host cell proteins during infection?

Investigating host-pathogen protein interactions involving CCA_00630 requires sophisticated methodological approaches:

• Proximity-dependent biotin labeling: Expressing CCA_00630 fused to BioID or APEX2 to identify proximal proteins in infected cells.

• Cross-linking mass spectrometry: Utilizing chemical cross-linkers followed by mass spectrometry to capture transient or weak interactions.

• Affinity purification coupled with mass spectrometry (AP-MS): Performing pull-downs with tagged CCA_00630 from infected cell lysates.

• Yeast two-hybrid screening against human cDNA libraries: Identifying potential binary interactions with host proteins.

• Validation using co-immunoprecipitation and co-localization studies: Confirming interactions in the context of infection.

• Functional validation through domain mapping and mutagenesis: Determining specific regions of CCA_00630 responsible for host protein interactions.

These approaches should be complemented by bioinformatic analyses to predict potential interaction interfaces based on the protein sequence and structure.

How can CCA_00630 be utilized in immunological studies of Chlamydophila infection?

While the search results don't specifically address immunological applications of CCA_00630, researchers can implement several methodological approaches based on studies of other Chlamydial proteins. The protein could be used to develop specific antibodies for immunodetection and localization studies within infected cells . Additionally, researchers could investigate whether CCA_00630 elicits an immune response during natural infection, and if so, characterize that response in terms of antibody production and T-cell activation. Drawing from immunization studies with other Chlamydial proteins like CPAF, researchers might examine whether recombinant CCA_00630 could serve as an antigen candidate in vaccine development approaches, particularly if the protein is found to be conserved across Chlamydia species and expressed during infection .

What considerations are important when designing experiments to study the expression patterns of CCA_00630 during different stages of the Chlamydial developmental cycle?

Designing rigorous experiments to study CCA_00630 expression throughout the Chlamydial developmental cycle requires careful methodological planning:

• Time-course sampling: Collect samples at multiple timepoints (0, 2, 6, 12, 24, 36, 48, and 72 hours post-infection) to capture expression across early, middle, and late stages of the developmental cycle.

• Quantitative transcript analysis: Implement RT-qPCR with carefully validated primers specific to CCA_00630 to measure transcript levels.

• Protein-level detection: Develop specific antibodies against CCA_00630 for Western blot and immunofluorescence microscopy to track protein production and localization.

• Single-cell analysis: Use fluorescence in situ hybridization or immunofluorescence microscopy to detect cell-to-cell variation in expression.

• Normalization strategy: Normalize expression data against established stage-specific markers and housekeeping genes to accurately interpret developmental regulation.

• Experimental controls: Include appropriate negative controls and positive controls using genes known to be expressed at specific developmental stages.

This methodological framework ensures reliable characterization of CCA_00630 expression patterns throughout the complex Chlamydial developmental cycle.

What approaches should be used to investigate whether CCA_00630 contains significant repeat sequences that might engage in recombination events?

To investigate potential repeat sequences in CCA_00630 that might participate in recombination, researchers should implement a multi-faceted bioinformatic and experimental approach:

• Computational sequence analysis: Apply specialized software tools like RepeatFinder or REPuter to identify large repeats (>23 nucleotides) capable of engaging in homologous recombination .

• SSR identification: Search for simple sequence repeats (SSRs), particularly homopolymeric tracts of G or C nucleotides, which are associated with hypervariability in Chlamydial genomes .

• Comparative genomics: Compare CCA_00630 sequences across multiple clinical isolates of C. caviae to identify polymorphic regions that might indicate ongoing recombination.

• PCR-based polymorphism detection: Design primers flanking potential variable regions and apply them to multiple strains to detect length or sequence polymorphisms.

• Experimental evolution studies: Subject C. caviae to multiple passages and sequence CCA_00630 to detect emergent variations.

This methodological approach follows established protocols used to identify recombination hotspots in other Chlamydial proteins, as documented in the literature .

How should researchers approach structure-function analysis of CCA_00630?

A comprehensive structure-function analysis of CCA_00630 requires an integrated methodological approach:

This methodological framework provides a systematic approach to correlating structural features with biological functions.

What methodological approaches could be used to determine if CCA_00630 has enzymatic activity?

Determining potential enzymatic activity of CCA_00630 requires a systematic approach beginning with bioinformatic analysis and progressing to biochemical characterization:

• Sequence motif analysis: Search for known catalytic motifs or active site signatures using databases like PROSITE or InterPro.

• Structural homology modeling: Identify structural similarity to known enzymes using tools like Phyre2 or I-TASSER.

• Activity screening: Test the purified protein against a panel of standard enzymatic activity assays covering major enzyme classes (hydrolases, transferases, oxidoreductases, etc.).

• Substrate specificity determination: If activity is detected, characterize substrate specificity using related compounds.

• Kinetic analysis: Determine enzyme kinetics parameters (Km, Vmax, kcat) for confirmed substrates.

• Cofactor requirements: Systematically test the effects of common enzyme cofactors (metal ions, nucleotides) on activity.

• Inhibitor studies: Use class-specific enzyme inhibitors to further characterize the type of enzymatic activity.

• Site-directed mutagenesis: Mutate predicted catalytic residues to confirm their role in the enzymatic mechanism.

This methodical approach provides a comprehensive assessment of potential enzymatic functions that might be encoded by this uncharacterized protein.

What are the key technical challenges in working with recombinant CCA_00630 and how can they be addressed?

Researchers working with recombinant CCA_00630 should anticipate several technical challenges:

• Protein solubility issues: If the protein forms inclusion bodies in E. coli, consider switching to yeast expression systems which may improve solubility . Alternatively, modify buffer conditions or add solubility enhancers such as low concentrations of non-ionic detergents.

• Protein stability concerns: The recommended addition of 5-50% glycerol to storage buffers helps maintain stability during freeze-thaw cycles . For proteins showing degradation, protease inhibitor cocktails should be included in all buffers.

• Functional activity retention: If the protein requires specific post-translational modifications for activity, expression in insect or mammalian cells may be necessary despite lower yields .

• Aggregation during storage: Implement dynamic light scattering or size exclusion chromatography quality control steps before and after storage to monitor aggregation states.

• Batch-to-batch variation: Establish robust quality control metrics including activity assays (once identified) to ensure consistency across preparations.

These technical considerations represent standard challenges in recombinant protein work, with solutions adapted specifically for CCA_00630 based on available information.

What quality control measures should be implemented when working with CCA_00630 in research settings?

A comprehensive quality control protocol for CCA_00630 should include:

• Purity assessment: While standard preparations achieve >85% purity by SDS-PAGE , researchers should implement multiple orthogonal methods including analytical size exclusion chromatography and mass spectrometry.

• Identity confirmation: Verify protein identity through western blotting with anti-tag antibodies and mass spectrometry peptide mapping against the expected sequence.

• Structural integrity evaluation: Use circular dichroism spectroscopy to assess secondary structure content and thermal stability, ensuring proper folding.

• Aggregation monitoring: Implement dynamic light scattering to detect protein aggregates that might affect functional studies.

• Endotoxin testing: For cell-based or in vivo applications, test for endotoxin contamination using LAL assays, particularly for preparations from E. coli.

• Batch record documentation: Maintain detailed records of expression conditions, purification parameters, and quality metrics for each preparation to track batch-to-batch consistency.

These quality control measures ensure experimental reproducibility and valid interpretation of results across different studies using this protein.

How can researchers optimize reconstitution protocols for CCA_00630 to maintain maximum biological activity?

Optimizing CCA_00630 reconstitution to preserve biological activity requires methodical attention to multiple parameters:

• Initial preparation: Follow recommended centrifugation of the vial before opening to collect all protein at the bottom .

• Reconstitution buffer selection: Use deionized sterile water for initial reconstitution to reach 0.1-1.0 mg/mL concentration , then consider buffer exchange to physiologically relevant buffers if needed for specific applications.

• Concentration optimization: Test a range of final protein concentrations to identify the optimal range that balances activity with stability.

• Additive screening: Systematically test protective additives beyond glycerol, such as low concentrations of reducing agents or carrier proteins if aggregation is observed.

• Temperature control: Perform all reconstitution steps at controlled temperatures (typically 4°C) to minimize thermal stress.

• Aliquoting strategy: Create single-use aliquots sized appropriately for planned experiments to eliminate freeze-thaw cycles .

• Activity validation: Develop and implement application-specific activity assays to confirm that the reconstituted protein maintains functionality.

This methodological approach to reconstitution optimization should be adapted based on the specific research applications planned for CCA_00630.

What comparative approaches can be used to analyze potential functional similarities between CCA_00630 and related proteins in other Chlamydial species?

To analyze functional similarities between CCA_00630 and related proteins across Chlamydial species, researchers should implement a systematic comparative approach:

• Homology-based identification: Use BLAST and HMM-based approaches to identify homologs of CCA_00630 in other Chlamydial species and related bacteria.

• Phylogenetic analysis: Construct phylogenetic trees to understand evolutionary relationships and potential functional divergence or conservation.

• Sequence conservation mapping: Calculate conservation scores for each residue and map them onto structural models to identify potentially functional regions.

• Synteny analysis: Examine the genomic context of CCA_00630 homologs across species to identify conserved gene neighborhoods that might suggest functional associations.

• Expression pattern comparison: Compare expression profiles of homologs during developmental cycles across different Chlamydial species.

• Complementation studies: Test functional interchangeability by expressing CCA_00630 in heterologous Chlamydial species with mutations in homologous genes.

• Domain architecture analysis: Compare domain organization and motifs across homologs to identify core functional elements versus species-specific adaptations.

This comparative framework provides insights into conserved functions while highlighting species-specific adaptations that might relate to host range or tissue tropism differences .

What are the recommended approaches for developing specific antibodies against CCA_00630 for research applications?

Developing high-quality antibodies against CCA_00630 requires a strategic methodological approach:

• Antigen design optimization:

  • Use full-length recombinant protein for polyclonal antibody development

  • Identify 2-3 antigenic peptides (15-20 amino acids) from hydrophilic, surface-exposed regions for monoclonal antibody development

  • Consider both N-terminal and C-terminal targeted antibodies to account for potential processing events

• Animal selection and immunization protocol:

  • For polyclonals: Rabbits typically provide good yields of high-affinity antibodies

  • For monoclonals: Consider mouse or rat hybridoma approaches

  • Implement prime-boost strategies with adjuvants appropriate for research antibodies

• Antibody validation requirements:

  • Western blot against recombinant protein and native protein from C. caviae lysates

  • Immunoprecipitation to confirm ability to recognize native protein

  • Immunofluorescence microscopy to validate recognition of the protein in fixed bacteria

  • Pre-absorption controls with recombinant protein to confirm specificity

  • Testing in knockout/knockdown backgrounds to confirm absence of signal

• Purification approach:

  • Affinity purification against the immunizing antigen

  • Consider cross-adsorption against lysates from related species to enhance specificity

This methodological framework ensures development of research-grade antibodies with validated specificity and utility across multiple applications.

What are the key knowledge gaps regarding CCA_00630 that represent priority areas for future research?

Several critical knowledge gaps regarding CCA_00630 warrant prioritized research attention:

• Functional characterization: The "UPF" (Uncharacterized Protein Family) designation indicates that the fundamental biological function remains unknown . Determining whether CCA_00630 has enzymatic activity, structural roles, or regulatory functions is a primary research priority.

• Expression patterns: Understanding when during the developmental cycle CCA_00630 is expressed, and whether expression varies under different environmental conditions or stresses, would provide insights into its biological context.

• Subcellular localization: Determining whether CCA_00630 localizes to specific bacterial compartments, the inclusion membrane, or is secreted into the host cytoplasm would suggest potential functional roles.

• Interaction partners: Identifying bacterial and host proteins that interact with CCA_00630 would provide significant clues about its biological role in Chlamydial biology.

• Structural characterization: Resolving the three-dimensional structure would facilitate structure-function analyses and potentially reveal functional domains not evident from sequence analysis alone.

• Genetic manipulation: Developing genetic systems to create knockout or knockdown strains would enable assessment of the protein's importance for bacterial growth, development, and virulence.

These interconnected research priorities would collectively advance understanding of this currently uncharacterized protein's role in Chlamydial biology.

How might comparative genomics approaches contribute to understanding the evolutionary significance of CCA_00630?

Comparative genomics approaches offer powerful insights into the evolutionary significance of CCA_00630 through several methodological strategies:

• Phylogenetic distribution analysis: Map the presence/absence pattern of CCA_00630 homologs across the bacterial phylogenetic tree to determine its evolutionary history and potential horizontal transfer events.

• Selection pressure analysis: Calculate dN/dS ratios across the coding sequence to identify regions under purifying selection (functionally constrained) versus diversifying selection (potentially involved in host adaptation).

• Synteny conservation assessment: Analyze the conservation of genomic context around CCA_00630 across species, as conserved gene neighborhoods often suggest functional relationships.

• Intraspecies variation studies: Compare CCA_00630 sequences across multiple strains of C. caviae to assess natural polymorphism levels, potentially revealing variable regions similar to the ppp family in C. pneumoniae .

• Recombination hotspot analysis: Apply computational methods to identify potential recombination sites within or surrounding CCA_00630, which might contribute to genetic variability .

• Co-evolution pattern detection: Identify proteins showing correlated evolutionary patterns with CCA_00630, suggesting functional relationships maintained through evolution.

These approaches could reveal whether CCA_00630 represents a core bacterial function or an adaptation specific to the Chlamydial lifestyle and host interactions.

What methodological approaches would be most appropriate for investigating potential roles of CCA_00630 in Chlamydial pathogenesis?

Investigating CCA_00630's potential roles in pathogenesis requires a multi-faceted methodological approach:

• Temporal expression analysis during infection: Use RT-qPCR and proteomics to determine if CCA_00630 expression correlates with key pathogenesis timepoints.

• Localization during infection: Employ immunofluorescence microscopy with specific antibodies to track CCA_00630 localization during the infection cycle, noting any translocation to the inclusion membrane or host cytosol.

• Host response studies: Expose host cells to purified recombinant CCA_00630 and measure inflammatory responses, signaling pathway activation, and transcriptional changes.

• Genetic manipulation approaches: Develop CCA_00630 knockout/knockdown strains or overexpression variants to assess impacts on infection efficiency, inclusion development, and host cell responses.

• Animal infection models: If genetic manipulation systems are available, compare wild-type and CCA_00630-modified strains in relevant animal models of infection.

• Host protein interaction screening: Identify host targets using approaches like BioID, yeast two-hybrid, or pull-down assays coupled with mass spectrometry.

• Comparative virulence analysis: Correlate naturally occurring variations in CCA_00630 across strains with differences in virulence phenotypes.

This integrated approach aligns with established methodologies for investigating the pathogenic roles of Chlamydial proteins, similar to studies conducted with CPAF .

How could structural biology approaches enhance understanding of CCA_00630 function?

Structural biology approaches would provide crucial insights into CCA_00630 function through several methodological avenues:

• High-resolution structure determination: X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy could reveal the protein's three-dimensional structure, potentially identifying functional domains not evident from sequence analysis.

• Structural homology analysis: Even in the absence of experimental structures, comparing predicted structural models with known protein structures could suggest functional similarities not detectable at the sequence level.

• Binding site identification: Computational prediction of binding pockets combined with experimental validation could identify potential interaction sites for substrates, cofactors, or protein partners.

• Conformational dynamics: Techniques like hydrogen-deuterium exchange mass spectrometry could reveal flexible regions that might undergo conformational changes during function.

• Structure-guided mutagenesis: Once key structural features are identified, targeted mutations can be designed to test their functional importance.

• Co-crystallization studies: Attempting crystallization with potential binding partners or substrates could capture functionally relevant complexes.

• Molecular dynamics simulations: Computational modeling of protein dynamics could suggest potential mechanisms and conformational changes relevant to function.

These structural approaches would complement biochemical and genetic studies to provide a more complete understanding of CCA_00630's molecular function.

What are the potential applications of CCA_00630 in developing diagnostic tools or therapeutic strategies for Chlamydial infections?

While maintaining focus on research applications rather than commercial aspects, several potential scientific applications of CCA_00630 in diagnostics and therapeutics warrant exploration:

• Serodiagnostic development: If CCA_00630 proves immunogenic during natural infection, assessing antibody responses could contribute to improved serological diagnostic tests with enhanced specificity.

• Molecular diagnostics: Primers targeting CCA_00630 could be incorporated into nucleic acid amplification tests, potentially offering species-specific detection capabilities.

• Vaccine antigen assessment: Following methodologies used for other Chlamydial proteins like CPAF, researchers could evaluate CCA_00630's potential as a component in multisubunit vaccine strategies by assessing immunogenicity and protective efficacy .

• Drug target evaluation: If functional characterization reveals enzymatic activity or essential roles in bacterial survival, CCA_00630 could be assessed as a potential target for antimicrobial development through structure-based drug design approaches.

• Protein-protein interaction disruption: If CCA_00630 interacts with host proteins during infection, these interfaces could represent targets for therapeutic intervention with small molecules or peptide mimetics.

These research directions would require thorough characterization of CCA_00630's biological function and significance in infection before translational applications could be pursued.