Recombinant Chicken Uncharacterized protein C17orf85 homolog (RCJMB04_3g9), partial

What is Recombinant Chicken Uncharacterized protein C17orf85 homolog?

The Recombinant Chicken Uncharacterized protein C17orf85 homolog (RCJMB04_3g9) is a protein that appears to function as a component of an alternative cap-binding complex in RNA processing, based on research on its human homolog. The human C17orf85 protein (also known as NCBP3) directly binds to the RNA cap structure depending on the N7-methyl group of the guanosine and interacts with NCBP1 to form an alternative cap-binding complex . This complex has redundant functions with the canonical CBC (Cap-Binding Complex) under steady-state conditions but becomes particularly important during antiviral responses .

What are the synonyms and alternative identifiers for this protein?

The protein is known by several names and identifiers in research literature and databases:

• Uncharacterized protein C17orf85 homolog

• ELG protein

• Chromosome 19 open reading frame, human C17orf85

• Gene names: C19H17ORF85, RCJMB04_3g9, C17orf85, HSA277841

What are the optimal storage conditions for maintaining protein stability?

For optimal stability and functionality, the recombinant protein should be stored at -20 degrees C for routine use. For long-term storage, maintaining the protein at -20 degrees C or -80 degrees C is recommended. Working aliquots can be stored at 4 degrees C for up to one week without significant degradation. It's important to note that repeated freezing and thawing cycles are not recommended as they can compromise protein structure and function .

What expression systems are used for producing recombinant chicken C17orf85 homolog?

The protein can be produced in multiple expression systems including E. Coli, Yeast, Baculovirus, or Mammalian Cell cultures . Each system offers different advantages for protein production:

The choice of expression system should be guided by the specific research requirements, particularly regarding protein folding and post-translational modifications .

How can researchers verify the identity and purity of recombinant chicken C17orf85 homolog?

Verification of identity and purity can be performed through multiple complementary approaches:

• Protein sequence analysis via mass spectrometry to confirm amino acid composition

• SDS-PAGE to assess purity (should be >90%)

• Western blotting with specific antibodies

• Immunocross-reactivity testing to confirm antigenic properties

• Molecular weight determination via size exclusion chromatography or mass spectrometry

These methods parallel the verification approaches used for other recombinant chicken proteins like recombinant chicken growth hormone (rcGH) .

What techniques are most effective for studying the RNA cap-binding properties of chicken C17orf85 homolog?

Based on studies of the human homolog, researchers should consider these methodological approaches:

• RNA electrophoretic mobility shift assays (EMSA) with differentially capped RNA substrates

• Fluorescence anisotropy measurements to quantify binding affinities

• Surface plasmon resonance (SPR) to determine binding kinetics

• RNA immunoprecipitation followed by sequencing (RIP-seq) to identify bound RNA species

• Structural analysis of protein-RNA complexes using X-ray crystallography or cryo-EM

These techniques would help establish whether the chicken homolog, like its human counterpart, specifically recognizes the N7-methyl group of the guanosine in the RNA cap structure .

How can researchers investigate the protein-protein interactions of chicken C17orf85 homolog?

To characterize potential interactions similar to the NCBP1-binding observed with the human homolog , researchers should employ:

• Co-immunoprecipitation assays with chicken NCBP1 and other potential binding partners

• Yeast two-hybrid screening to identify novel interaction partners

• Proximity-based labeling methods such as BioID or APEX

• Fluorescence resonance energy transfer (FRET) for in vivo interaction studies

• Quantitative binding assays such as isothermal titration calorimetry (ITC)

Such studies would reveal whether the chicken C17orf85 homolog forms an alternative cap-binding complex analogous to the human system .

How might researchers design experiments to elucidate the role of chicken C17orf85 homolog in antiviral responses?

Given the established role of the human homolog in antiviral defense , researchers should consider:

• RNA interference (RNAi) or CRISPR-Cas9 knockout studies in chicken cell lines followed by viral challenge

• Overexpression studies to assess protective effects against avian viruses

• Transcriptomic analysis comparing wild-type and C17orf85-deficient cells during viral infection

• Immunoprecipitation studies during viral infection to identify virus-specific interactions

• In vivo studies using targeted approaches to modulate C17orf85 expression in chickens

The experimental designs should include appropriate controls and time-course analyses to distinguish between direct antiviral functions and secondary effects .

What bioinformatic approaches can predict the structural and functional properties of chicken C17orf85 homolog?

Comprehensive bioinformatic analysis should include:

These computational approaches would provide testable hypotheses about structure-function relationships in the chicken C17orf85 homolog.

What are common challenges in obtaining functionally active recombinant chicken C17orf85 homolog?

Researchers should be aware of several potential challenges:

• Protein misfolding due to inappropriate expression system selection

• Loss of activity during purification processes

• Aggregation in storage buffers lacking appropriate stabilizers like glycerol

• Batch-to-batch variation in activity

• Insufficient removal of contaminants affecting functional assays

To address these challenges, researchers should optimize expression conditions, include stabilizing agents like glycerol in storage buffers , and perform rigorous activity testing between production batches.

How can researchers distinguish between direct and indirect effects in functional studies?

To differentiate direct from indirect effects when studying chicken C17orf85 homolog function:

• Design rescue experiments with wild-type versus mutant protein versions

• Employ time-course studies to establish temporal relationships between events

• Use domain mapping and site-directed mutagenesis to identify critical functional regions

• Compare acute (short-term) versus chronic (long-term) depletion effects

• Utilize in vitro reconstitution assays with purified components

Such approaches are particularly important when investigating complex phenotypes like antiviral responses, where multiple pathways may be affected .

How does the chicken C17orf85 homolog compare functionally to its mammalian counterparts?

While detailed comparative studies between chicken and mammalian C17orf85 homologs are not explicitly described in the available data, researchers should approach this question by:

• Conducting cross-species complementation assays

• Comparing binding affinities for RNA caps and protein partners

• Analyzing expression patterns across tissues in both species

• Examining responses to identical viral challenges

• Evaluating structural conservation of key functional domains

The human homolog functions as part of an alternative cap-binding complex with NCBP1 and plays a role in antiviral responses , providing a framework for comparative studies with the chicken homolog.

What insights can be gained from studying the evolution of cap-binding complexes across species?

Evolutionary analysis of cap-binding complexes including C17orf85 homologs can reveal:

• Conservation patterns indicating functional importance of specific domains

• Species-specific adaptations potentially related to pathogen pressure

• Co-evolution with viral antagonists that target RNA processing

• Lineage-specific expansion or contraction of cap-binding protein families

• Structural innovations that might confer novel functionalities in different species

Such evolutionary perspectives could provide insights into the adaptation of RNA processing mechanisms across vertebrate lineages.

How does research on chicken C17orf85 homolog contribute to understanding avian immune responses?

Research on this protein contributes to avian immunology by:

• Elucidating avian-specific post-transcriptional regulation mechanisms

• Providing insights into bird-specific adaptations for viral defense

• Identifying potential targets for enhancing immune responses in poultry

• Revealing evolutionary conservation and divergence in innate immunity pathways

• Contributing to our understanding of species-specific susceptibilities to pathogens

The homolog's potential role in antiviral responses, similar to its human counterpart , makes it particularly relevant for understanding avian immunity to economically important viral diseases.

What methodologies can link C17orf85 homolog function to transcriptome-wide effects in chicken cells?

To establish connections between this protein and global gene expression patterns, researchers should consider:

• RNA-seq analysis comparing wild-type and C17orf85-depleted chicken cells

• CLIP-seq (Crosslinking and immunoprecipitation followed by sequencing) to map direct binding sites

• Ribosome profiling to assess translational impacts

• Integration of transcriptomic and proteomic datasets

• Network analysis to identify regulatory hubs affected by C17orf85 perturbation

These approaches parallel methodologies used in other avian transcriptional profiling studies but would be specifically focused on cap-dependent RNA processing events.

What emerging technologies could advance our understanding of chicken C17orf85 homolog function?

Several cutting-edge approaches hold promise:

• Cryo-electron microscopy for structural determination of protein-RNA complexes

• Single-molecule imaging to visualize cap-binding dynamics in live cells

• Targeted protein degradation approaches for acute protein depletion

• CRISPR activation/interference for nuanced modulation of expression

• Integrative multi-omics approaches combining transcriptomics, proteomics, and metabolomics

These technologies would provide more comprehensive insights into the protein's function than traditional biochemical or genetic approaches alone.

How might understanding C17orf85 homolog function contribute to avian disease research?

Advanced knowledge of this protein could impact disease research through:

• Identification of novel antiviral targets for veterinary interventions

• Development of biomarkers for viral susceptibility in poultry

• Engineering of disease-resistant chicken lines through targeted genetic approaches

• Improved understanding of host-pathogen co-evolution in avian species

• Creation of research tools for studying post-transcriptional regulation in avian disease models

This research direction aligns with studies examining host responses to avian pathogens, such as the transcriptional profiling of chicken macrophages during APEC infection .