Recombinant Xenopus laevis Coiled-coil domain-containing protein 93 (ccdc93), partial

What is the predicted role of ccdc93 in Xenopus laevis cellular function?

Based on comparative studies with other organisms, ccdc93 likely functions as a component of the retriever complex, which is involved in recycling integral plasma membrane proteins . In Arabidopsis, CCDC93 has been shown to function in endosomal trafficking pathways essential for cellular growth, suggesting a conserved role across species . The coiled-coil domains characteristic of this protein facilitate protein-protein interactions critical for membrane trafficking networks.

For researchers investigating ccdc93 function, initial characterization should include co-immunoprecipitation experiments coupled with mass spectrometry to identify interaction partners within the Xenopus cellular context. Confocal microscopy with fluorescently tagged ccdc93 constructs can determine subcellular localization and potential co-localization with known endosomal markers.

What expression systems are optimal for producing functional recombinant Xenopus ccdc93?

Both yeast and E. coli expression systems have been successfully employed for recombinant production of Xenopus laevis ccdc93 . Each system offers distinct advantages depending on research requirements.

E. coli expression (CSB-EP753585XBE) provides high yield and is suitable for structural studies, though proper folding of complex domains may be challenging . When selecting this system, consider using specialized E. coli strains designed for expressing eukaryotic proteins and optimizing induction conditions to enhance soluble protein production.

Yeast expression (CSB-YP753585XBE) often provides better post-translational modifications and protein folding for eukaryotic proteins . For functional assays requiring properly folded protein, this system may be preferable despite typically lower yields compared to bacterial systems.

What are the advantages of biotinylated ccdc93 for interaction studies?

Biotinylated recombinant ccdc93 (CSB-EP753585XBE-B) produced using AviTag-BirA technology offers significant advantages for interaction studies . This method creates a covalent amide linkage between biotin and a specific lysine residue in the AviTag peptide, providing consistent site-specific biotinylation.

The primary methodological advantages include:

• High-affinity, stable binding to streptavidin matrices for pulldown assays

• Oriented immobilization for surface plasmon resonance studies

• Reduced interference with protein function compared to random chemical biotinylation

• Enhanced sensitivity for detecting low-abundance interaction partners

For researchers investigating ccdc93 protein interactions, the biotinylated variant enables more rigorous quantitative binding studies and more effective purification of intact protein complexes.

How can CRISPR-Cas9 gene editing be optimized for studying ccdc93 function in Xenopus?

Xenopus provides unique advantages for CRISPR-Cas9 approaches, including the ability to analyze gene function in transheterozygotes of the F0 generation only hours after application . For ccdc93 studies, CRISPR-Cas9 offers powerful options for loss-of-function analysis.

Methodological considerations include:

• gRNA design should target early exons of ccdc93 to ensure complete protein disruption

• Delivery can be achieved through microinjection at the one-cell stage

• Analysis can begin in transheterozygotes of the F0 generation

• Targeted mosaics can be created to study tissue-specific functions

Given the likely role of ccdc93 in membrane trafficking, CRISPR-mediated knockout should be complemented with detailed phenotypic analysis of endosomal compartments using fluorescent markers.

What approaches are effective for studying ccdc93's role in cellular metabolism during Xenopus development?

Recent studies have highlighted the importance of metabolic regulation in Xenopus development and regeneration processes . Though ccdc93's specific role in metabolism is not yet established, its potential involvement in membrane protein recycling suggests it may influence metabolic signaling pathways.

To investigate this connection, researchers should consider:

• Metabolomic profiling comparing wild-type and ccdc93-deficient embryos

• Analysis of glycolytic pathway activity, as glycolytic shifts have been observed during Xenopus regeneration

• Mitochondrial function assessment using fluorescent probes for membrane potential

• Investigation of potential interactions between ccdc93 and metabolism-regulating receptors

These approaches can reveal whether ccdc93 influences the metabolic shifts observed during development and regeneration in Xenopus laevis.

How might ccdc93 contribute to the remarkable regenerative capacity of Xenopus laevis?

Xenopus laevis exhibits significant regenerative abilities, particularly in larval stages, with neural stem precursor cells (NSPCs) playing a crucial role in spinal cord regeneration . While direct evidence linking ccdc93 to this process is lacking, its potential role in membrane protein trafficking suggests it could influence regenerative signaling pathways.

To explore this connection, researchers should consider the following experimental approaches:

• Temporal expression analysis of ccdc93 during regeneration processes

• Loss-of-function studies using CRISPR-Cas9 or morpholinos to assess effects on regenerative capacity

• Investigation of ccdc93 interaction with key regenerative signaling receptors

• Analysis of whether the transient glycolytic shift observed during regeneration correlates with changes in ccdc93 expression or activity

These studies could reveal previously unidentified roles for ccdc93 in the remarkable regenerative processes characteristic of Xenopus laevis.

What genetic interaction studies would best elucidate ccdc93 function in Xenopus?

In Arabidopsis, genetic interaction studies have revealed that CCDC93 shares a genetic interaction with the VTI13-dependent trafficking pathway to the vacuole . Similar genetic approaches in Xenopus could provide valuable insights into ccdc93 function.

Methodological approach for genetic interaction studies:

• Generate ccdc93 mutant lines using CRISPR-Cas9

• Identify potential genetic interactors based on known membrane trafficking components

• Create double mutant lines through crossing

• Perform detailed phenotypic analysis, focusing on:

  • Developmental timing and morphogenesis

  • Cellular trafficking processes

  • Organelle morphology and distribution

  • Response to regeneration-inducing injuries

These genetic interaction studies would help position ccdc93 within the complex cellular trafficking networks in Xenopus cells.

How can researchers determine if ccdc93 function is conserved between Xenopus and mammals?

Conservation analysis is critical for understanding fundamental protein functions and for translating findings between model systems. Determining functional conservation of ccdc93 between Xenopus and mammals requires multiple complementary approaches.

Methodological strategies include:

• Sequence and structural comparative analysis using bioinformatics tools

• Cross-species rescue experiments:

  • Express mammalian ccdc93 in Xenopus ccdc93-deficient embryos

  • Assess functional complementation through phenotypic rescue

• Domain swap experiments to identify functionally critical regions

• Comparative interactome analysis using consistent immunoprecipitation protocols

These approaches would establish the degree of functional conservation and highlight any species-specific adaptations in ccdc93 function.

What methodological considerations are important when comparing ccdc93 interactomes across species?

The interactome of a protein can vary significantly between species despite sequence conservation. For meaningful cross-species comparison of ccdc93 interaction partners, several methodological considerations are crucial:

• Experimental consistency:

  • Use identical tagging strategies (N- or C-terminal tags)

  • Apply the same affinity purification protocols

  • Employ comparable cell types or developmental stages

• Data analysis standardization:

  • Use consistent statistical thresholds for identifying interactions

  • Apply identical filtering criteria for background binding

  • Normalize interaction strengths appropriately

• Validation approaches:

  • Confirm key interactions with reciprocal co-immunoprecipitation

  • Verify co-localization using microscopy

  • Test functional relevance through targeted disruption

Such methodologically rigorous comparative studies would reveal conserved and divergent aspects of ccdc93 function across species.