Recombinant Human Uncharacterized protein C3orf71 (C3orf71)

What expression systems are most suitable for recombinant C3orf71 production?

Recombinant Uncharacterized protein C3orf71 can be successfully expressed in multiple host systems, with E. coli and yeast offering optimal yields and shorter turnaround times for basic structural studies . For applications requiring post-translational modifications, insect cells with baculovirus or mammalian expression systems are recommended despite their lower yield, as these systems provide many of the post-translational modifications necessary for correct protein folding or retained activity . When selecting an expression system, researchers should consider their specific experimental requirements:

• E. coli expression: Ideal for high-yield production of the protein for basic structural studies, antibody production, or when post-translational modifications are not critical

• Yeast expression: Offers a balance between yield and some eukaryotic post-translational modifications

• Insect/mammalian cell expression: Essential when studying protein function that may depend on specific post-translational modifications

When expressing C3orf71 in E. coli, the protein has been successfully produced as a full-length construct (amino acids 1-290) with an N-terminal His-tag .

What are the optimal storage conditions for recombinant C3orf71?

Recombinant C3orf71 protein is typically provided as a lyophilized powder and requires careful handling for maximum stability and activity . The following storage protocol is recommended based on standard approaches for similar uncharacterized proteins:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) and aliquot for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as they can significantly decrease protein activity

• For working solutions, store aliquots at 4°C for up to one week

How can I verify the purity and integrity of expressed C3orf71?

The quality control of recombinant C3orf71 should follow standard protein biochemistry approaches. Commercially available recombinant C3orf71 typically shows greater than 90% purity as determined by SDS-PAGE . Researchers should implement the following verification methods:

• SDS-PAGE analysis: Run purified protein on a 10-12% gel to verify the expected molecular weight and purity

• Western blot: Use anti-His antibodies (for His-tagged versions) or specific antibodies against C3orf71 if available

• Mass spectrometry: Perform peptide mass fingerprinting to confirm protein identity

• Size exclusion chromatography: Assess protein aggregation state and homogeneity

These complementary approaches provide comprehensive quality control for recombinant C3orf71 preparations.

How does C3orf71 compare to the better-characterized C2orf71?

While C3orf71 remains largely uncharacterized, parallels with C2orf71 may provide valuable insights:

Although these proteins share the nomenclature pattern of being named after their chromosomal locations, their sequences, sizes, and potentially their functions appear distinct. Researchers should exercise caution when making functional inferences between these proteins.

What methodologies are recommended for studying C3orf71 subcellular localization?

Understanding the subcellular localization of C3orf71 is crucial for elucidating its function. Based on approaches used for related proteins like C2orf71, the following methodologies are recommended:

• Immunofluorescence microscopy:

  • Express tagged versions (GFP, FLAG, or His) in relevant cell lines

  • Use organelle-specific markers to determine colocalization patterns

  • Include confocal microscopy for high-resolution localization

• Cellular fractionation:

  • Separate nuclear, cytoplasmic, membrane, and organelle fractions

  • Detect C3orf71 distribution using Western blotting

  • Compare distribution patterns under different cellular conditions

• Proximity labeling approaches:

  • Use BioID or APEX2 fusion proteins to identify proximal interacting partners

  • Map the protein to specific subcellular compartments based on identified neighbors

Drawing from C2orf71 research, where the protein was found to localize to primary cilia in cultured cells , investigators might specifically examine whether C3orf71 shows similar localization patterns or associations with specific subcellular structures.

What experimental approaches can be used to investigate the function of uncharacterized C3orf71?

The functional analysis of uncharacterized proteins like C3orf71 requires a multi-faceted approach:

• Gene knockdown/knockout studies:

  • Use siRNA or CRISPR-Cas9 to reduce or eliminate C3orf71 expression

  • Analyze resulting phenotypes at cellular and molecular levels

  • Consider model organisms for in vivo studies, similar to the zebrafish approach used for C2orf71

• Overexpression studies:

  • Express wild-type and tagged versions in relevant cell lines

  • Assess effects on cellular processes, morphology, and viability

  • Compare with known phenotypes of related proteins

• Interactome analysis:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Use yeast two-hybrid or mammalian two-hybrid systems

  • Implement proximity-based labeling techniques (BioID, APEX)

• Transcriptome/proteome analysis:

  • Compare gene expression and protein profiles in cells with and without C3orf71

  • Identify pathways or processes affected by C3orf71 manipulation

The zebrafish model system proved particularly valuable for C2orf71 functional studies, where knockdown resulted in visual defects . Similar approaches could be considered for C3orf71 depending on its expression pattern and suspected functions.

How can protein degradation pathways be studied for C3orf71 variants?

Protein stability and degradation are crucial aspects of protein function. Based on studies of C2orf71, where mutations led to proteasomal degradation , the following approaches can be applied to C3orf71:

• Protein stability assays:

  • Express wild-type and variant forms of C3orf71 in cell culture

  • Measure protein half-life using cycloheximide chase assays

  • Compare expression levels of variants via Western blotting

• Degradation pathway inhibition:

  • Use specific inhibitors like MG115 (proteasome only) and MG132 (proteasome and cathepsin K)

  • Assess protein levels before and after inhibitor treatment

  • Identify which degradation pathways are involved based on inhibitor effects

• Ubiquitination analysis:

  • Immunoprecipitate C3orf71 and probe for ubiquitin

  • Co-express with tagged ubiquitin to visualize ubiquitination patterns

  • Identify specific lysine residues that undergo ubiquitination

This methodology mirrors the approach used in C2orf71 studies, where researchers found that both MG115 and MG132 inhibitors rescued expression of the mutant protein, indicating proteasomal degradation as the primary mechanism .

What tissue expression analysis should be performed to understand C3orf71 biology?

Determining the tissue expression pattern of C3orf71 is essential for understanding its biological context:

• RT-PCR and qPCR analysis:

  • Design specific primers targeting C3orf71 mRNA

  • Screen a panel of human tissues for expression

  • Use developmental time courses in model organisms to determine temporal expression patterns

• In situ hybridization:

  • Create RNA probes for C3orf71

  • Perform hybridization on tissue sections to localize expression

  • Compare with markers of specific cell types

• Public database mining:

  • Analyze RNA-seq and microarray data from repositories like GTEx, TCGA, and GEO

  • Compare expression patterns across tissues, developmental stages, and disease states

  • Look for correlation with functionally related genes

For C2orf71, such expression analyses revealed that it was highly specific to retinal photoreceptor cells . Similar analyses for C3orf71 would help determine its tissue specificity and provide clues to its function.

How might C3orf71 be involved in disease processes?

While direct disease associations for C3orf71 are not well established in the provided search results, researchers can employ several strategies to investigate potential disease relevance:

• Genetic association studies:

  • Analyze genomic data from disease cohorts for variants in C3orf71

  • Perform targeted sequencing in patient populations with relevant phenotypes

  • Screen for copy number variations affecting the C3orf71 locus

• Expression analysis in disease states:

  • Compare C3orf71 expression between normal and diseased tissues

  • Look for correlations between expression levels and disease progression

  • Analyze single-cell RNA-seq data to identify cell-specific expression changes

• Functional studies in disease models:

  • Manipulate C3orf71 expression in disease-relevant cell or animal models

  • Assess effects on disease phenotypes or pathogenic mechanisms

  • Test whether restoration of normal C3orf71 function rescues disease phenotypes

Given that C3orf71 is also known as ARIH2OS (Ariadne-2 homolog opposite strand protein) , researchers should investigate potential functional relationships with ARIH2, which is involved in ubiquitination processes and cellular regulation.

What are the challenges in developing specific antibodies against C3orf71?

Developing specific antibodies against uncharacterized proteins like C3orf71 presents several challenges:

• Epitope selection considerations:

  • Perform bioinformatic analyses to identify unique, surface-exposed regions

  • Avoid regions with high similarity to other proteins

  • Select multiple epitopes from different protein regions to increase success probability

• Validation strategies:

  • Use tagged recombinant C3orf71 as positive control

  • Include knockout or knockdown samples as negative controls

  • Perform peptide competition assays to confirm specificity

  • Test antibodies using multiple techniques (Western blot, IP, IF, IHC)

• Cross-reactivity assessment:

  • Test antibodies against related proteins

  • Evaluate antibody performance across species if evolutionary conservation is relevant

  • Perform mass spectrometry validation of immunoprecipitated proteins

Researchers should be particularly careful to distinguish between C3orf71 and potential homologs or related proteins to ensure antibody specificity.

What structural biology approaches are suitable for an uncharacterized protein like C3orf71?

Understanding the three-dimensional structure of C3orf71 would provide significant insights into its function:

• Computational structure prediction:

  • Use AlphaFold2 or RoseTTAFold to generate structural models

  • Perform molecular dynamics simulations to assess structural stability

  • Identify potential functional sites through structural analysis

• Experimental structure determination:

  • Express and purify protein domains suitable for structural studies

  • Screen crystallization conditions for X-ray crystallography

  • Consider NMR spectroscopy for smaller domains or flexible regions

  • Utilize cryo-EM for larger complexes if C3orf71 forms stable associations

• Hybrid approaches:

  • Combine low-resolution experimental data with computational modeling

  • Use cross-linking mass spectrometry to obtain distance constraints

  • Validate structural predictions with mutational analyses

These approaches would require high-quality recombinant protein, which can be produced in E. coli with an N-terminal His-tag as described in the available product information .

What strategies can overcome expression challenges for C3orf71?

Researchers may encounter difficulties expressing C3orf71, particularly in prokaryotic systems. The following strategies can help overcome common challenges:

• Optimization of expression conditions:

  • Test multiple expression strains (BL21, Rosetta, Arctic Express)

  • Vary induction conditions (temperature, IPTG concentration, duration)

  • Consider auto-induction media for gradual protein expression

• Solubility enhancement approaches:

  • Express with solubility-enhancing tags (MBP, SUMO, TrxA)

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Optimize buffer conditions during cell lysis and purification

• Domain-based expression:

  • Identify and express individual domains rather than full-length protein

  • Design constructs based on bioinformatic predictions of domain boundaries

  • Test multiple constructs with varying N- and C-terminal boundaries

The reported successful expression of full-length C3orf71 (1-290) with an N-terminal His-tag in E. coli suggests that prokaryotic expression is feasible , though optimization may be required for specific experimental needs.

How can potentially conflicting data about C3orf71 function be reconciled?

When investigating uncharacterized proteins like C3orf71, researchers may encounter conflicting experimental results. The following approaches can help resolve such contradictions:

• Systematic comparison of experimental conditions:

  • Document all experimental variables (cell types, expression methods, tags)

  • Replicate studies under identical conditions

  • Test whether specific conditions trigger different protein behaviors

• Multiple methodological approaches:

  • Verify findings using complementary techniques

  • Combine in vitro, cellular, and in vivo approaches

  • Consider both gain-of-function and loss-of-function studies

• Context-dependent function assessment:

  • Investigate whether the protein functions differently in various cellular contexts

  • Test function under different physiological stresses

  • Consider potential tissue-specific roles

This systematic approach acknowledges that proteins may have context-dependent functions and helps build a more complete understanding of C3orf71 biology.