Recombinant Danio rerio UPF0697 protein C8orf40 homolog (zgc:114040)

What is the amino acid sequence and structural composition of Recombinant Danio rerio UPF0697 protein C8orf40 homolog?

The Recombinant Danio rerio UPF0697 protein C8orf40 homolog (zgc:114040) presents a complete amino acid sequence as follows: MGGYGVMADEESLDYSVHEAWNEATNVYLLVILVSFALLMYARKNKRKIMRIFTLPPTVGSSSEPNFYDSLQKVRLRQQLEMYSLARKYDQQQSQSESVQLSME . This 104-amino acid sequence represents the full-length protein as indicated in the product specifications. The protein is classified under the UPF0697 protein family, which typically consists of uncharacterized protein families awaiting functional annotation.

The primary structure suggests potential transmembrane domains, particularly in the region containing the hydrophobic amino acid stretch "VYLLVILVSFALLMY," which is characteristic of membrane-associated proteins. This region may be critical for the protein's localization and function within cellular compartments. The presence of charged amino acids (lysine, arginine) in proximity to this region further suggests potential interaction sites with other cellular components.

What is known about the functional role of UPF0697 protein C8orf40 homolog in Danio rerio?

While specific functional characterization of the zgc:114040 gene product remains limited, this protein represents a zebrafish homolog of the human C8orf40 (Chromosome 8 open reading frame 40) protein . Functional annotation of this protein remains under the UPF classification (Uncharacterized Protein Family), indicating that detailed molecular functions await further characterization through experimental studies.

Based on sequence homology and conservation patterns across species, several hypotheses can be proposed regarding its function. The protein likely plays a role in fundamental cellular processes conserved between humans and zebrafish. The presence of potential membrane-spanning domains suggests it may function in cellular compartmentalization, membrane organization, or transmembrane signaling. Researchers should consider experimental designs that investigate protein-protein interactions, subcellular localization, and phenotypic effects of gene knockdown to elucidate its biological role.

How does the expression pattern of zgc:114040 vary during zebrafish development?

Current research data on the developmental expression pattern of zgc:114040 is limited. Researchers investigating this aspect should consider employing techniques such as whole-mount in situ hybridization (WISH) to visualize spatial and temporal expression patterns during embryonic and larval development. This approach would provide valuable insights into potential tissue-specific functions.

Comparative analysis with expression patterns of the human ortholog C8orf40 might provide additional context for understanding evolutionary conservation of function. Temporal expression profiling through quantitative PCR at different developmental stages would complement spatial data obtained through WISH. Such comprehensive expression analysis would establish a foundation for functional studies by identifying critical developmental timepoints and tissues for targeted experimentation.

What are the optimal conditions for using Recombinant Danio rerio UPF0697 protein in ELISA-based experimental systems?

For optimal ELISA applications using Recombinant Danio rerio UPF0697 protein C8orf40 homolog, researchers should carefully consider buffer composition, protein concentration, and detection systems. The protein is supplied in a Tris-based buffer with 50% glycerol , which may require dilution to reduce glycerol concentration in the final assay.

The following protocol considerations are recommended:

• Working concentration: Begin with 1-5 μg/ml for coating ELISA plates, with optimization based on specific antibody sensitivity.

• Buffer conditions: Phosphate-buffered saline (PBS) at pH 7.4 is suitable for most applications.

• Blocking solution: 3-5% BSA or 5% non-fat milk in PBS with 0.05% Tween-20 to minimize non-specific binding.

• Detection system: Selection between direct or sandwich ELISA formats depends on available antibodies specific to the UPF0697 protein.

• Temperature considerations: All incubations should be performed at room temperature (20-25°C) unless specified otherwise.

Researchers should also validate the functional integrity of the protein after thawing by running a small aliquot on SDS-PAGE prior to ELISA applications.

How can molecular dynamics simulation be applied to study the structural stability of UPF0697 protein?

Molecular dynamics (MD) simulation represents a powerful approach for investigating the structural stability and conformational changes of the UPF0697 protein C8orf40 homolog under various physiological conditions. Drawing from methodologies applied to other zebrafish proteins like MC1R , researchers can implement similar approaches for UPF0697 protein analysis.

The methodology would involve:

As demonstrated with the MC1R protein, temperature-dependent stability analysis at both 300K and 350K can provide insights into thermostability and conformational flexibility . For UPF0697 protein, such analysis would be particularly relevant for identifying stable domains suitable for functional studies or antibody development.

What approaches can be used to investigate protein-protein interactions involving UPF0697 protein?

Understanding the interactome of UPF0697 protein C8orf40 homolog would significantly advance knowledge of its biological function. Several complementary experimental approaches are recommended:

• Pull-down assays using the recombinant protein as bait, followed by mass spectrometry to identify binding partners.

• Yeast two-hybrid screening against a zebrafish cDNA library to detect direct protein interactions.

• Proximity-dependent biotin identification (BioID) by expressing the protein fused to a biotin ligase in zebrafish cells.

• Co-immunoprecipitation studies using antibodies against the recombinant protein in zebrafish tissue lysates.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding affinities with predicted interaction partners.

Computational approaches should complement experimental methods, including:

• Prediction of interaction interfaces based on structural models

• Conservation analysis to identify functionally important regions

• Molecular docking simulations with potential binding partners

Integration of these approaches would establish a functional network context for UPF0697 protein, potentially revealing signaling pathways or cellular processes in which it participates.

What storage and handling procedures should be followed to maintain protein stability?

Proper storage and handling of Recombinant Danio rerio UPF0697 protein C8orf40 homolog are critical for maintaining functional integrity across experiments. According to product specifications, the following guidelines should be strictly observed:

• Long-term storage: Maintain at -20°C, with -80°C recommended for extended storage periods beyond six months .

• Working aliquots: Store at 4°C for up to one week to minimize freeze-thaw cycles .

• Buffer conditions: The protein is supplied in a Tris-based buffer with 50% glycerol, optimized for stability .

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity .

• Temperature transitions: Allow frozen aliquots to thaw completely at 4°C before bringing to room temperature for experimental use.

For experiments requiring dilution, researchers should consider:

• Using buffers with stabilizing agents such as 0.1% BSA

• Preparing fresh dilutions for each experiment when possible

• Validating protein integrity via activity assays or structural analysis after storage periods

These precautions will ensure experimental reproducibility and maintain the functional characteristics of the recombinant protein across studies.

How can researchers validate the structural integrity of UPF0697 protein after expression and purification?

Validating the structural integrity of recombinant UPF0697 protein is essential before proceeding with functional studies. A multi-technique approach is recommended:

• SDS-PAGE analysis: To confirm molecular weight (approximately 11-12 kDa based on the 104 amino acid sequence) and purity.

• Western blotting: Using antibodies against the protein or tag to verify identity.

• Circular dichroism (CD) spectroscopy: To assess secondary structure content and folding state.

• Thermal shift assays: To determine stability under different buffer conditions.

• Limited proteolysis: To probe for correctly folded domains resistant to protease digestion.

• Mass spectrometry: For accurate mass determination and potential post-translational modifications.

For advanced structural validation:

• Dynamic light scattering (DLS) to check for aggregation

• Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Nuclear magnetic resonance (NMR) spectroscopy for proteins that can be isotopically labeled

These analytical approaches should be employed as quality control measures before proceeding to functional studies, ensuring that experimental outcomes reflect the properties of properly folded protein.

What expression systems are most effective for producing functional UPF0697 protein?

The selection of an appropriate expression system for Recombinant Danio rerio UPF0697 protein C8orf40 homolog depends on research objectives and downstream applications. Based on available information and practices for similar zebrafish proteins, several expression systems merit consideration:

• E. coli expression system:

  • Advantages: Rapid growth, high yield, cost-effective

  • Limitations: Potential issues with proper folding of membrane-associated domains

  • Recommendation: Suitable for applications requiring denatured protein or where refolding protocols are established

• Yeast expression system:

  • Advantages: Eukaryotic post-translational modifications, proper folding of complex proteins

  • Limitations: Lower yield compared to bacterial systems

  • Recommendation: Preferable for functional studies requiring native conformation

• Baculovirus expression system:

  • Advantages: High-level expression of eukaryotic proteins with proper folding

  • Limitations: More complex setup, longer production time

  • Recommendation: Optimal for proteins requiring specific post-translational modifications

• Mammalian cell expression:

  • Advantages: Most physiologically relevant post-translational modifications

  • Limitations: Highest cost, lower yield

  • Recommendation: Consider for applications demanding highest functional fidelity

The tag selection also impacts purification efficiency and protein functionality. Options include:

• His-tag for metal affinity chromatography

• GST-tag for improved solubility

• Avi-tag for site-specific biotinylation as observed with other zebrafish proteins

The final choice should balance yield requirements with the need for functional integrity in downstream applications.

What functional evolutionary insights can be gained from studying the zebrafish UPF0697 protein?

The zebrafish model offers unique advantages for evolutionary functional studies of UPF0697 protein. By analyzing this protein in Danio rerio, researchers can address several key evolutionary questions:

• Functional conservation: The degree to which the protein's biological role has been preserved across evolutionary distance from teleosts to mammals.

• Expression pattern conservation: Whether developmental and tissue-specific expression profiles show similar patterns between zebrafish and mammals.

• Interactome evolution: How protein-protein interaction networks involving UPF0697 protein have evolved and whether core interactions are maintained.

Using approaches such as:

• Reciprocal genetic rescue experiments (can human C8orf40 replace zebrafish homolog function?)

• Comparative expression studies throughout development

• Parallel interaction studies in both zebrafish and human cells

These investigations would provide insights into how fundamental cellular processes involving this protein have evolved, potentially revealing both conserved essential functions and species-specific adaptations. This evolutionary perspective is particularly valuable for understanding the basic biological role of this uncharacterized protein family.

How can CRISPR-Cas9 genome editing be employed to study UPF0697 protein function in zebrafish?

CRISPR-Cas9 genome editing offers powerful approaches for functional characterization of the UPF0697 protein C8orf40 homolog in vivo. A comprehensive experimental design would include:

• Knockout strategy:

  • Design of guide RNAs targeting critical exonic regions of zgc:114040

  • Generation of frameshift mutations leading to complete loss of function

  • Phenotypic analysis across developmental stages and adult tissues

  • Molecular phenotyping through transcriptomics and proteomics

• Knockin modifications:

  • Introduction of fluorescent protein tags for live imaging of protein localization

  • Creation of specific point mutations to test functional hypotheses

  • Introduction of human sequence to test cross-species functional conservation

• Conditional approaches:

  • Implementation of inducible systems to control gene expression temporally

  • Tissue-specific knockout using Cre-lox systems to address tissue autonomy

• High-throughput phenotypic screening:

  • Behavioral assays to detect neurological phenotypes

  • Developmental timing analysis

  • Morphological assessments at cellular and organismal levels

This comprehensive CRISPR-based approach would provide insights into UPF0697 protein function that cannot be obtained through in vitro studies alone, establishing the physiological relevance of this protein family in vertebrate development and homeostasis.