Recombinant Danio rerio Coiled-coil domain-containing protein 43 (ccdc43)

What is Recombinant Danio rerio Coiled-coil Domain-containing Protein 43 (CCDC43)?

Recombinant Danio rerio CCDC43 is a protein derived from zebrafish (Danio rerio, also known as Brachydanio rerio) that contains coiled-coil domains. It is commercially available as a recombinant protein tagged with histidine (His tag) for research purposes. The protein typically encompasses amino acids 1-213 of the full-length zebrafish CCDC43 sequence and is primarily used in biochemical and molecular biology research applications . The coiled-coil domain structure suggests potential protein-protein interaction capabilities, which is characteristic of this protein family. The recombinant version is produced in expression systems such as yeast to facilitate its purification and subsequent use in experimental settings.

How is recombinant zebrafish CCDC43 typically expressed and purified?

Recombinant zebrafish CCDC43 is typically expressed using eukaryotic expression systems, with yeast being a common and economically efficient choice for this particular protein . The expression approach generally follows these methodological steps:

• Expression System Selection: Yeast is preferred for CCDC43 expression due to its cost-effectiveness as a eukaryotic system and its ability to perform post-translational modifications that might be important for protein functionality .

• Vector Construction: The CCDC43 gene sequence (coding for AA 1-213) is cloned into an expression vector that includes a His-tag coding sequence to facilitate purification.

• Protein Expression: Transformed yeast cells express the recombinant protein under controlled conditions.

• Purification Process: The protein is typically purified using affinity chromatography, taking advantage of the His-tag which binds to nickel or cobalt resin.

• Quality Control: The purity is assessed, with commercial preparations typically achieving >90% purity .

Alternative expression systems include E. coli for prokaryotic expression or mammalian cell lines for higher eukaryotic expression, though these may result in differences in cost, yield, and post-translational modifications . The choice of expression system should be considered carefully based on the specific research requirements.

What are the common applications for recombinant CCDC43 in zebrafish research?

Recombinant zebrafish CCDC43 has several research applications in academic settings, primarily centered around:

• Enzyme-Linked Immunosorbent Assay (ELISA): The recombinant protein can be used in ELISA applications, typically for the development and validation of antibodies against CCDC43 or for studying protein-protein interactions .

• Western Blotting: Commercial preparations of recombinant CCDC43 are suitable for Western blotting applications, allowing researchers to validate antibody specificity or study protein expression patterns .

• Functional Studies: Though not explicitly detailed in the search results, recombinant CCDC43 could potentially be used in binding assays to identify interacting partners, which is particularly relevant given the protein's coiled-coil domains that often mediate protein-protein interactions.

• Gene Expression Network Analysis: In broader zebrafish research contexts, proteins like CCDC43 may be studied as part of gene co-expression networks to understand their roles in response to chemical exposures or developmental processes .

These applications provide researchers with tools to investigate the biological roles and molecular mechanisms involving CCDC43 in zebrafish, which serves as an important model organism for vertebrate development and disease.

How can researchers optimize experimental design when working with zebrafish CCDC43?

When designing experiments involving recombinant zebrafish CCDC43, researchers should consider several methodological approaches to optimize results:

• Protein Reconstitution: For lyophilized preparations, proper reconstitution is crucial. The recommended protocol involves centrifuging the vial at 10,000 rpm for 1 minute, followed by gentle reconstitution at 200 μg/mL in sterile distilled water through gentle pipetting 2-3 times, avoiding vortexing which can damage the protein .

• Buffer Considerations: The protein is typically lyophilized from a solution containing 10 mM Hepes, 500 mM NaCl with 5% trehalose at pH 7.4 . Researchers should consider these buffer conditions when designing experiments to maintain protein stability.

• Working Dilution Determination: The optimal working dilution should be determined empirically for each specific application, as effectiveness may vary between experimental contexts .

• Storage Conditions: Proper storage is essential for maintaining protein activity. Lyophilized protein can be stored at -20°C for up to 12 months, while reconstituted protein should be stored at 2-8°C and used within 1 month under sterile conditions .

• Controls: When using zebrafish CCDC43 in experiments, appropriate positive and negative controls should be included to validate results and ensure specificity of observed effects.

By considering these factors, researchers can enhance experimental reproducibility and increase the likelihood of obtaining meaningful results when studying CCDC43 in zebrafish models.

What is known about CCDC43 function in zebrafish gene networks?

While the specific function of CCDC43 in zebrafish is not thoroughly characterized in the provided search results, insights can be drawn from broader gene network studies in zebrafish:

• Gene Co-expression Networks: Zebrafish research has utilized gene co-expression network analysis to understand how genes respond to chemical exposures. These networks can reveal functional relationships between genes like CCDC43 and other components of cellular pathways .

• Chemical Response Patterns: Gene co-expression studies have shown that different chemical exposures can induce distinct patterns of gene expression in zebrafish. For example, flame retardant chemicals (FRCs) and aryl hydrocarbon receptor 2 (AHR2) activators induce different transcriptomic responses, with FRCs affecting a broader range of processes including neurobehavioral pathways .

• Module Association: In gene co-expression networks, genes are often grouped into functional modules. Understanding which module CCDC43 associates with could provide insights into its potential biological processes and molecular functions.

• Developmental Context: Given the importance of zebrafish as a model for developmental biology, CCDC43 may play roles in specific developmental processes, though this would require targeted studies to elucidate.

Future research specifically focusing on CCDC43 within these gene networks could provide more definitive insights into its function in zebrafish biology and development.

How might CCDC43 relate to disease models in zebrafish?

While direct evidence linking CCDC43 to disease models in zebrafish is not presented in the search results, several potential connections can be hypothesized based on related research:

• Ciliary Function: Some research suggests connections between coiled-coil domain-containing proteins and ciliary function . Primary Ciliary Dyskinesia (PCD) involves mutations in genes affecting ciliary structure and function, with zebrafish models helping to understand the systemic importance of cilia . If CCDC43 plays a role in ciliary function, it could potentially be relevant to cilia-related disorders.

• Cancer Pathways: Zebrafish has been established as a model for studying cancer genes. Research has shown that many ribosomal protein genes act as haploinsufficient tumor suppressors in zebrafish . If CCDC43 interacts with or affects ribosomal protein function, it could potentially influence cancer-related pathways.

• Chemical Toxicity: Gene co-expression network analysis in zebrafish has revealed how chemicals alter transcriptomic responses . CCDC43 might be part of response pathways to chemical exposures, potentially relevant to toxicology studies and environmental health research.

• Developmental Disorders: Given the importance of proper protein expression and function during development, alterations in CCDC43 could potentially affect developmental processes in zebrafish, serving as a model for human developmental disorders.

These potential connections remain speculative until specific studies investigating CCDC43 in disease contexts are conducted. Zebrafish models offer an excellent platform for such investigations due to their genetic tractability and vertebrate physiology.

What techniques are most effective for studying CCDC43 function in zebrafish?

Several experimental techniques can be effectively applied to study CCDC43 function in zebrafish:

• Gene Expression Analysis: Techniques like qPCR, RNA-seq, or in situ hybridization can be used to characterize the spatial and temporal expression patterns of CCDC43 during zebrafish development or in response to experimental conditions.

• Gene Co-expression Network Analysis: Methods such as GENIE3 (a random forest method) have been successfully applied in zebrafish to build gene co-expression networks that can reveal functional relationships between genes . This approach could place CCDC43 within broader functional contexts.

• Morpholino Knockdown or CRISPR/Cas9 Gene Editing: These techniques can be used to reduce or eliminate CCDC43 expression in zebrafish embryos, allowing researchers to observe resulting phenotypes and infer protein function.

• Protein Localization Studies: Immunohistochemistry or fluorescent protein tagging can determine the subcellular localization of CCDC43, providing clues to its function.

• Protein-Protein Interaction Assays: Techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity labeling methods can identify proteins that interact with CCDC43, helping to elucidate its functional networks.

• Functional Rescue Experiments: Following knockdown or knockout, reintroduction of wild-type or mutant forms of CCDC43 can help define essential functional domains of the protein.

These methodological approaches provide complementary information that, when integrated, can offer comprehensive insights into CCDC43 function in zebrafish development and physiology.

How can researchers effectively use recombinant CCDC43 in immunological applications?

For effective use of recombinant CCDC43 in immunological applications such as ELISA and Western blotting, researchers should consider the following methodological guidelines:

• ELISA Protocol Optimization:

  • Use the recombinant protein at appropriate concentrations, starting with manufacturer recommendations (if available) and optimizing through titration experiments.

  • Ensure proper blocking to reduce background signal.

  • Include suitable controls, such as unrelated proteins with similar tags to control for tag-specific binding.

  • Consider the buffer components used for reconstitution and dilution, as they may affect antibody binding.

• Western Blotting Protocol:

  • The recombinant CCDC43 protein can serve as a positive control when detecting endogenous CCDC43 in zebrafish samples.

  • Optimal protein loading should be determined empirically, but starting with 50-100 ng of recombinant protein is reasonable.

  • When using anti-His antibodies to detect the tagged protein, appropriate controls should be included to distinguish tag-specific from protein-specific signals.

• Antibody Development and Validation:

  • Recombinant CCDC43 can be used as an immunogen for antibody production or for validating commercially available antibodies.

  • Validation should include testing antibody specificity using both the recombinant protein and zebrafish tissue samples where CCDC43 is expressed.

• Storage and Handling Considerations:

  • Follow manufacturer guidelines for storage (typically -20°C for lyophilized protein) .

  • Once reconstituted, use the protein within the recommended time frame (typically 1 month when stored at 2-8°C) .

  • Avoid repeated freeze-thaw cycles that can lead to protein degradation.

These methodological considerations can help ensure reliable and reproducible results when using recombinant CCDC43 in immunological applications for zebrafish research.

How does zebrafish CCDC43 compare to orthologs in other species?

A comparative analysis of CCDC43 across species can provide evolutionary insights and functional predictions:

• Sequence Conservation: While specific comparative data for CCDC43 is not provided in the search results, coiled-coil domain-containing proteins often show conservation of functional domains across species while allowing for some sequence divergence in less critical regions.

• Functional Conservation: The degree to which CCDC43 function is conserved across species remains to be thoroughly investigated. Comparative studies could reveal whether zebrafish CCDC43 functions similarly to its mammalian counterparts or has evolved species-specific roles.

• Expression Patterns: Comparing the tissue-specific and developmental expression patterns of CCDC43 across species could provide clues about conserved and divergent functions.

• Interacting Partners: Identification and comparison of CCDC43-interacting proteins across species could reveal conserved functional networks or species-specific interactions.

• Disease Associations: In humans, mutations in some coiled-coil domain-containing proteins are associated with specific diseases. Comparative studies could reveal whether zebrafish CCDC43 might serve as a model for understanding human CCDC43-related conditions, if any exist.

Systematic comparative analysis of CCDC43 across multiple model organisms could enhance our understanding of its fundamental biological roles and evolutionary significance.

What are promising future research directions for CCDC43 in zebrafish?

Based on the available information and broader research contexts, several promising future research directions for CCDC43 in zebrafish can be identified:

• Functional Characterization: Comprehensive studies using gene knockdown or knockout approaches could elucidate the specific biological functions of CCDC43 in zebrafish development, physiology, and disease responses.

• Integration into Gene Co-expression Networks: Incorporating CCDC43 into existing zebrafish gene co-expression networks could reveal its functional associations with other genes and biological pathways . This could be particularly informative in the context of chemical exposures or developmental processes.

• Potential Role in Ciliary Function: Given the importance of ciliary function in zebrafish and the potential connection of coiled-coil domain-containing proteins to ciliary structures, investigating whether CCDC43 plays a role in cilia formation or function could be valuable .

• Chemical Response Studies: Examining how CCDC43 expression or function changes in response to various chemical exposures could provide insights into its role in toxicological responses and stress adaptation .

• Protein Interaction Network Mapping: Identifying proteins that interact with CCDC43 could reveal its molecular mechanisms and cellular functions.

• Comparative Studies with Human CCDC43: Research comparing zebrafish and human CCDC43 could establish zebrafish as a model for understanding CCDC43 function in human health and disease.

These research directions could significantly advance our understanding of CCDC43 biology and potentially reveal new insights into fundamental cellular processes and disease mechanisms.