Recombinant Chlamydophila caviae UPF0109 protein CCA_00022 (CCA_00022)

What is Chlamydophila caviae UPF0109 protein CCA_00022?

Recombinant Chlamydophila caviae UPF0109 protein CCA_00022 is a full-length protein derived from the genome of Chlamydophila caviae (strain GPIC). It belongs to the UPF0109 protein family, designated in protein databases with the UniProt accession number Q824W6 . This protein is recombinantly expressed in E. coli expression systems to generate sufficient quantities for research applications . The protein consists of 78 amino acids corresponding to the complete native sequence without additional mutations or variations. CCA_00022 is part of the catalog of annotated genes identified during the genome sequencing of Chlamydophila caviae, making it one of the 1,009 genes identified in this obligate intracellular bacterial pathogen . As a member of the UPF0109 family, it represents a protein with a currently uncharacterized function, designated by the "UPF" (Uncharacterized Protein Family) nomenclature.

What are the recommended protocols for reconstitution and storage of recombinant CCA_00022?

The reconstitution of recombinant CCA_00022 requires careful handling to maintain protein integrity and activity. Prior to opening, it is recommended that the vial containing lyophilized protein be briefly centrifuged to ensure all contents settle at the bottom . The protein should be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For long-term storage, it is crucial to add glycerol to a final concentration of 5-50%, with 50% being the standard recommendation for optimal preservation . Following reconstitution, the protein solution should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly compromise protein stability and activity. For storage, recombinant CCA_00022 in liquid form maintains stability for approximately 6 months when kept at -20°C or -80°C, while the lyophilized form can remain stable for up to 12 months at the same temperatures . For short-term use, working aliquots may be stored at 4°C for up to one week, though this is not recommended for longer periods . These storage conditions are particularly important for maintaining the structural integrity of the protein, especially considering that repeated freezing and thawing can lead to protein denaturation and loss of functional properties.

What expression systems and purification methods are most effective for CCA_00022?

The expression of recombinant CCA_00022 is most commonly achieved using E. coli expression systems, which offer high yield and relatively straightforward purification protocols . Based on related recombinant protein expression methods, such as those used for Chlamydophila felis CF0218, expression typically involves cloning the target gene into a suitable vector, such as the GST fusion protein expression vector pGEX-6P-1 . The expression protocol would typically involve transforming E. coli BL21 or similar strains with the recombinant construct, followed by induction of protein expression using IPTG (isopropyl β-d-1-thiogalactopyranoside) . For optimal expression, culture conditions generally involve growth at 30-37°C, with induction occurring during the logarithmic growth phase . Following expression, purification typically employs affinity chromatography techniques, taking advantage of the fusion tag incorporated into the recombinant construct. For GST-tagged proteins, purification using glutathione Sepharose affinity chromatography is effective, while His-tagged constructs would utilize Ni-NTA or similar metal-affinity resins . After affinity purification, the fusion tag can be removed if necessary using specific proteases, such as PreScission protease for GST tags . Final purification often includes size exclusion chromatography or ion exchange chromatography to achieve high purity, with the commercial product specification indicating a purity of >85% as determined by SDS-PAGE analysis .

How can researchers verify the identity and purity of recombinant CCA_00022?

Verification of recombinant CCA_00022 identity and purity requires a multi-faceted analytical approach. SDS-PAGE analysis provides the primary method for assessing protein purity, with commercial preparations typically achieving >85% purity as verified by this technique . For molecular weight confirmation, the expected size of the full-length 78-amino acid protein can be compared against standard markers. More definitive identification requires Western blot analysis using antibodies specific to CCA_00022 or to fusion tags if present. Mass spectrometry offers the most precise method for identity confirmation, providing accurate molecular weight determination and, through peptide mapping, sequence verification. For structural integrity assessment, circular dichroism spectroscopy can evaluate secondary structure elements. Functional verification may be challenging for proteins of unknown function like UPF0109 family members, but comparative analysis with related proteins could provide insights. Additionally, researchers should consider conducting tests for endotoxin contamination, especially critical for immunological studies, and verify the absence of proteolytic degradation through time-course stability studies under experimental conditions. Collectively, these analytical approaches ensure that the recombinant protein meets the necessary quality standards for reliable research applications.

What is known about the structure and function of UPF0109 family proteins?

The UPF0109 protein family, to which CCA_00022 belongs, remains largely uncharacterized in terms of specific function, as indicated by the "UPF" (Uncharacterized Protein Family) designation . The primary sequence of CCA_00022 consists of 78 amino acids: MKDFLAYIIK NLVDRPEEVH IKEVQGTHTI IYELTVAKPD IGKIIGKEGR TIKAIRTLLV SVASRNNVKV SLEIMEDK . Structural analysis suggests that UPF0109 proteins likely adopt a specific fold, though detailed three-dimensional structures determined by X-ray crystallography or NMR spectroscopy are currently limited in the literature. Based on sequence analysis, these proteins may contain transmembrane domains or membrane-associated regions, similar to other chlamydial proteins like the TMH family protein CF0218 from Chlamydophila felis . Functional predictions based on conserved domains and sequence homology suggest potential roles in cellular processes, possibly related to bacterial physiology or host-pathogen interactions. The conservation of CCA_00022 across chlamydial species indicates biological significance, despite the current lack of characterized function . Understanding the structural properties of this protein family requires computational prediction methods such as secondary structure prediction, transmembrane domain analysis, and homology modeling based on structurally characterized proteins with similar sequences.

What post-translational modifications might affect CCA_00022 function?

Post-translational modifications (PTMs) potentially affecting CCA_00022 function remain largely uncharacterized, requiring predictive analysis based on sequence features. The 78-amino acid sequence (MKDFLAYIIK NLVDRPEEVH IKEVQGTHTI IYELTVAKPD IGKIIGKEGR TIKAIRTLLV SVASRNNVKV SLEIMEDK) contains several lysine, serine, threonine, and tyrosine residues that could serve as modification sites . Potential PTMs might include phosphorylation of serine, threonine, or tyrosine residues, which often regulate protein activity, localization, or interactions. Lysine residues could undergo acetylation, ubiquitination, or SUMOylation, affecting protein stability or function. For bacterial proteins, methylation, glycosylation, and lipidation also represent possible modifications with functional consequences. Computational prediction tools can identify potential modification sites based on consensus sequences, but experimental verification through mass spectrometry or specific antibodies against modified peptides would be necessary for confirmation. When using recombinant CCA_00022 expressed in E. coli, researchers should note that the bacterial expression system may not reproduce the native PTM pattern found in Chlamydophila caviae, potentially affecting functional studies. Comparative analysis of native CCA_00022 isolated from C. caviae with the recombinant version could reveal functionally significant modifications, though such studies present technical challenges due to the obligate intracellular nature of chlamydial bacteria.

How is recombinant CCA_00022 utilized in immunological studies?

Recombinant CCA_00022 serves as a valuable tool in immunological studies investigating Chlamydophila caviae infections and immune responses. The purified protein can be used as an antigen for generating polyclonal or monoclonal antibodies, following protocols similar to those employed for related chlamydial proteins like CF0218 . Such antibodies enable immunological detection of native CCA_00022 in infected cells or tissues, facilitating studies of protein expression, localization, and potential role during infection. Immunization studies in animal models can assess the immunogenicity of CCA_00022 and determine whether antibodies against this protein contribute to protective immunity against C. caviae infection. Western blot analysis using recombinant CCA_00022 can evaluate cross-reactivity with antibodies raised against other chlamydial species, providing insights into antigenic conservation and potential for cross-protection . Additionally, the recombinant protein can be utilized in T-cell stimulation assays to characterize cell-mediated immune responses, complementing humoral immunity studies. ELISA-based serological assays incorporating recombinant CCA_00022 may assist in epidemiological surveys of C. caviae prevalence or in distinguishing C. caviae infections from other chlamydial infections. For vaccines development research, CCA_00022 could be evaluated as a potential subunit vaccine component or as a carrier protein for delivering specific immunogenic epitopes.

What protein-protein interaction studies can be performed using CCA_00022?

Protein-protein interaction studies using recombinant CCA_00022 can provide valuable insights into its functional networks and biological role. Pull-down assays represent a fundamental approach, where GST-tagged or His-tagged CCA_00022 can be immobilized on appropriate affinity resins and used to capture interacting partners from C. caviae lysates . Co-immunoprecipitation studies using anti-CCA_00022 antibodies can identify protein complexes containing CCA_00022 in more native conditions. For quantitative interaction analysis, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can determine binding affinities and kinetics between CCA_00022 and candidate interacting proteins. Yeast two-hybrid screens offer an unbiased approach to identify novel interaction partners, though validation through orthogonal methods is essential. For in situ visualization of protein interactions, proximity ligation assays or FRET-based approaches can be employed in infected cell models. Crosslinking mass spectrometry (XL-MS) provides detailed information about interaction interfaces by identifying residues in close proximity between CCA_00022 and its binding partners. Bacterial two-hybrid systems may be particularly suitable for studying interactions within the bacterial intracellular environment. Computational predictions of protein-protein interactions based on structural modeling can guide experimental designs by identifying potential interaction surfaces. These methodologies collectively contribute to mapping the interactome of CCA_00022 and understanding its functional context within C. caviae biology.

How can CCA_00022 be used in studying C. caviae pathogenesis?

Recombinant CCA_00022 offers multiple approaches for investigating Chlamydophila caviae pathogenesis. Expression analysis using RT-PCR or qPCR with CCA_00022-specific primers, similar to methods used for other chlamydial genes, can determine temporal expression patterns during the developmental cycle, indicating potential stage-specific functions . Immunofluorescence microscopy using anti-CCA_00022 antibodies can reveal the protein's localization within infected cells, suggesting whether it associates with inclusion membranes, bacterial surfaces, or remains within the bacterial cytoplasm. Functional studies employing recombinant CCA_00022 in host cell cultures can assess direct effects on cellular processes, signaling pathways, or cytokine responses. RNA interference or CRISPR-based approaches targeting host factors potentially interacting with CCA_00022 can identify cellular components required for its function. Comparative studies examining CCA_00022 homologs across chlamydial species with different host tropisms or disease manifestations may provide evolutionary insights into pathoadaptation. In animal models of C. caviae infection, such as guinea pig inclusion conjunctivitis, immunization with recombinant CCA_00022 can evaluate its role in protective immunity. Structural studies of CCA_00022, facilitated by the availability of purified recombinant protein, can identify potential binding sites for host factors or therapeutic compounds. These multifaceted approaches collectively contribute to understanding CCA_00022's potential role in the pathogenic mechanisms of C. caviae.

What are common challenges in working with recombinant CCA_00022?

Researchers working with recombinant CCA_00022 may encounter several technical challenges requiring specific troubleshooting approaches. Protein solubility issues can arise during expression and purification, potentially necessitating optimization of buffer conditions, temperature, or the use of solubility-enhancing fusion tags beyond standard GST or His tags . Protein stability concerns may emerge, particularly during storage or experimental manipulations, requiring careful attention to recommended storage conditions (-20°C/-80°C with glycerol) and avoidance of repeated freeze-thaw cycles . Contamination with bacterial endotoxins can confound immunological studies, necessitating endotoxin removal steps during purification, especially for in vivo applications. Proper folding of the recombinant protein compared to the native form presents another consideration, as E. coli expression systems may not reproduce all post-translational modifications or disulfide bond formations present in the native chlamydial environment . Antibody specificity issues can arise in immunological applications, requiring careful validation of antibodies against recombinant CCA_00022 and controls to exclude cross-reactivity with other bacterial or host proteins . Functional activity assessment presents particular challenges for proteins of unknown function like CCA_00022, requiring creative approaches based on putative functional predictions or comparative studies with characterized homologs. These methodological considerations demand careful experimental design and appropriate controls to ensure reliable and reproducible results when working with this recombinant protein.

How can antibody cross-reactivity issues with CCA_00022 be addressed?

Addressing antibody cross-reactivity issues requires systematic validation and appropriate experimental controls. When generating antibodies against recombinant CCA_00022, purify the immunizing antigen to high homogeneity (>85% purity by SDS-PAGE) to minimize response to contaminating proteins . Pre-adsorb antisera against E. coli lysates if the recombinant protein was expressed in bacterial systems, eliminating antibodies recognizing residual bacterial proteins. Perform cross-reactivity testing against homologous proteins from related Chlamydia species to characterize species-specificity, similar to the testing performed with C. felis CF0218 and antisera against C. trachomatis and C. psittaci . Employ Western blot analysis to evaluate antibody specificity against both recombinant CCA_00022 and native protein in C. caviae-infected cell lysates, confirming recognition of the correct molecular weight target . For immunofluorescence or immunohistochemistry applications, include uninfected controls and competitive inhibition controls using excess recombinant CCA_00022 to demonstrate binding specificity. Consider epitope mapping to identify specific regions recognized by polyclonal antibodies, potentially allowing selection of less conserved epitopes for improved specificity. For monoclonal antibodies, screen multiple clones to identify those with highest specificity for CCA_00022 versus homologs. These systematic approaches to antibody validation minimize cross-reactivity concerns and enhance the reliability of immunological studies involving CCA_00022.

How does CCA_00022 fit into the evolutionary history of chlamydial species?

The evolutionary position of CCA_00022 within the context of chlamydial species provides insights into its biological significance. As one of the 798 genes conserved across all sequenced chlamydial genomes, CCA_00022 likely evolved early in chlamydial lineage development and maintains important functions preserved through selective pressure . Phylogenetic analysis based on sequence comparisons with homologs in C. pneumoniae, C. trachomatis, and C. muridarum can reveal evolutionary relationships and rates of sequence divergence . Unlike proteins encoded within the replication termination region (RTR), which shows substantial variation between chlamydial species and often contains species-specific genes, CCA_00022 appears to be part of the more stable core genome . The conservation pattern contrasts with the 68 genes unique to C. caviae that lack orthologs in other completed chlamydial genomes, suggesting different evolutionary trajectories for core versus accessory genes . Sequence conservation analysis across homologs can identify highly conserved residues potentially critical for function, versus more variable regions that may confer species-specific adaptations. Comparison with distant bacterial homologs beyond the Chlamydiaceae family, if present, could provide broader evolutionary context regarding the ancestral origins of this protein family. This evolutionary perspective enhances understanding of CCA_00022's biological role and its potential as a target for broad-spectrum interventions across chlamydial species.

What are the key differences between recombinant and native CCA_00022?

Understanding the differences between recombinant and native CCA_00022 is crucial for interpreting experimental results. Recombinant CCA_00022 produced in E. coli expression systems may lack post-translational modifications present in the native protein within C. caviae, potentially affecting function, localization, or interaction capabilities . The recombinant protein typically includes additional elements not present in the native form, such as affinity tags (His, GST) for purification, which may influence structural properties or create steric hindrance in interaction studies, necessitating tag removal in some applications . Folding differences may arise from the distinct cellular environments of E. coli versus C. caviae, potentially affecting secondary and tertiary structure formation, especially for disulfide bonds or other modifications requiring specialized cellular machinery . While the native CCA_00022 exists in the context of the chlamydial intracellular environment, potentially as part of multi-protein complexes, the recombinant form is studied in isolation, removing contextual factors that might influence function. The purity profile differs significantly—recombinant CCA_00022 is typically purified to >85% homogeneity, whereas native protein exists among numerous other chlamydial and host cell proteins . Additionally, the recombinant protein is available in precisely quantifiable amounts, enabling controlled experimental conditions, whereas native CCA_00022 expression levels during infection may vary with developmental stage and cannot be easily manipulated. These distinctions must be considered when extrapolating findings from recombinant protein studies to biological processes in C. caviae infection.

How do UPF0109 family proteins compare across bacterial species?

The UPF0109 protein family, represented by CCA_00022 in Chlamydophila caviae, exhibits varying patterns of conservation across bacterial species. Comparative genomic analysis reveals that UPF0109 family members are present in multiple bacterial phyla, suggesting ancient evolutionary origins predating the divergence of major bacterial lineages. Within the Chlamydiaceae family, UPF0109 proteins show relatively high sequence conservation, consistent with the finding that CCA_00022 is among the 798 genes conserved across all sequenced chlamydial genomes . This conservation pattern contrasts with the substantial genomic variation observed in the replication termination region (RTR) of chlamydial genomes, which contains many species-specific genes . Sequence alignment of UPF0109 family members across diverse bacteria reveals conserved motifs potentially critical for structural integrity or function, alongside more variable regions that may confer species-specific adaptations. Domain architecture analysis indicates that UPF0109 proteins typically function as standalone units rather than as parts of larger multi-domain proteins, suggesting discrete functional roles. The small size of these proteins (78 amino acids for CCA_00022) is relatively consistent across species, indicating functional constraints on protein length . While structural data for most UPF0109 family members remains limited, computational predictions suggest conservation of secondary structure elements across bacterial species, supporting functional similarity despite sequence divergence. This comparative analysis provides a framework for understanding the broader biological significance of CCA_00022 beyond its specific role in C. caviae.

Future research directions and methodological recommendations

Future research on CCA_00022 should prioritize functional characterization through complementary approaches. Gene expression analysis using RT-PCR or RNA-Seq can determine temporal expression patterns during the chlamydial developmental cycle, providing initial functional insights . Subcellular localization studies using fluorescently tagged protein or immunofluorescence microscopy with specific antibodies can reveal association with particular cellular structures. Protein-protein interaction studies, including pull-down assays, co-immunoprecipitation, and crosslinking approaches, should be pursued to identify binding partners and functional networks . Structural biology techniques, including X-ray crystallography and NMR spectroscopy, would provide valuable three-dimensional information to guide functional predictions. For functional analysis in the challenging context of obligate intracellular bacteria, heterologous expression systems or cell-free protein synthesis methods may be necessary. Comparative functional studies with homologs from other bacterial species could leverage existing knowledge to illuminate CCA_00022 function. CRISPR interference or similar genetic approaches adapted for chlamydial systems could assess the impact of CCA_00022 depletion on bacterial viability and function. Methodologically, researchers should carefully consider the limitations of recombinant proteins versus native forms, employing appropriate controls to address potential differences . Interdisciplinary approaches combining structural biology, proteomics, genetic manipulation, and computational modeling will likely be necessary to fully characterize this protein of unknown function, potentially revealing new aspects of chlamydial biology and identifying novel therapeutic targets.

Table 1: Key Properties of Recombinant Chlamydophila caviae UPF0109 protein CCA_00022

Table 2: Comparative Genomic Context of Chlamydophila caviae Within the Chlamydiaceae Family