Recombinant Human Uncharacterized protein C2orf74 (C2orf74)

What is the genomic structure of C2orf74 and how can researchers effectively analyze it?

C2orf74, also known as LOC339804, is a protein-encoding gene located on chromosome 2 at position 2p15. The gene spans 19,713 base pairs on the plus strand, beginning at position 61,145,116 and ending at 61,164,828 in the human genome, containing 8 exons in total . Neighboring genes include KIAA841, LOC105374759, LOC105374758, LOC339803, AHSA2P, USP34, and SNORA70B.

For genomic analysis, researchers should:

• Utilize genome browsers (UCSC, Ensembl) to visualize exon-intron structure

• Analyze promoter regions using tools like JASPAR or TRANSFAC to identify transcription factor binding sites

• Design primers spanning exon-exon junctions for specific cDNA amplification

• Consider chromatin immunoprecipitation sequencing (ChIP-seq) to identify regulatory elements

Gene neighborhood analysis can provide functional insights through association with better-characterized genes in the region, particularly when investigating potential co-regulation patterns or shared regulatory elements.

What are the known protein isoforms of C2orf74 and how should researchers approach isoform-specific studies?

C2orf74 has six validated mRNA transcript variants created through alternative splicing, giving rise to two distinct protein isoforms :

For isoform-specific research approaches:

• Design isoform-specific PCR primers or RT-qPCR assays targeting unique exon junctions

• Create expression constructs for each isoform to study functional differences

• Develop antibodies targeting isoform-specific regions

• When analyzing expression data from public databases, distinguish between transcripts

The extended form (194 aa) is particularly noteworthy as it utilizes a 5' in-frame start codon, though it's not formally recognized as a separate isoform by NCBI . Researchers should specify which isoform they're using in publications, as functional differences may exist between these variants.

How conserved is C2orf74 across species and what methodological approaches reveal evolutionary insights?

C2orf74 has orthologs in approximately 135 different species, primarily placental mammals and some marsupials . Sequence homology analysis reveals varying degrees of conservation:

• 95% identity with Cynomolgus monkey

• 58% identity with Pig

• 50% identity with Mouse

This conservation pattern suggests the protein may have mammal-specific functions that evolved relatively recently, with higher conservation among primates indicating possible specialized functions.

For evolutionary analyses, researchers should:

• Perform multiple sequence alignments (Clustal Omega, MUSCLE) to identify conserved domains

• Construct phylogenetic trees to visualize evolutionary relationships

• Apply selective pressure analysis (dN/dS ratios) to identify regions under positive or purifying selection

• Examine synteny across species to understand evolutionary history of the locus

• Consider compensatory evolution patterns in potential binding partners

The moderate conservation level (50% with mouse) suggests functional constraints but allows for species-specific adaptations, making cross-species functional studies particularly informative.

What methods are most effective for predicting structural features of C2orf74?

As an uncharacterized protein, C2orf74's structure remains to be experimentally determined. The core sequence has been identified as:
TKKVPCTDANGGVDCAAAKVVTSNPEDHERILMQVMNLNVPMRPGILVQRQSKEVLATPLENRRDMEAEEENQINEKQEPENAGETGQEEDDGLQKIHTSVTRTPSVVESQKRPLKGVTFSREVIVVDLGNEYPTPRSYT

For structural prediction, researchers should employ a multi-tiered approach:

• Apply bioinformatic tools (InterPro, SMART, Pfam) to identify potential domains

• Use secondary structure prediction algorithms (PSIPRED, JPred)

• Analyze the protein with AlphaFold2 or RoseTTAFold for tertiary structure prediction

• Create hydrophobicity plots to identify potential membrane-associated regions

• Apply disorder prediction tools (PONDR, IUPred) to identify intrinsically disordered regions

• Consider experimental approaches like limited proteolysis coupled with mass spectrometry

The isoelectric point of 6.5 indicates a slightly acidic protein, which may influence buffer selection for experimental work. The presence of two cysteine residues in the core sequence suggests potential disulfide bond formation that could be critical for proper folding.

What expression systems are optimal for producing recombinant C2orf74 and what optimization strategies are recommended?

Escherichia coli has been successfully used as an expression system for recombinant human C2orf74, as evidenced by commercially available proteins . The molecular weight of the expressed protein is approximately 18 kDa .

For expression system selection and optimization:

• E. coli systems: Use BL21(DE3) or Rosetta strains for high-level expression

  • Optimize induction temperature (16°C, 25°C, 37°C)

  • Test different IPTG concentrations (0.1-1.0 mM)

  • Consider auto-induction media for higher cell densities

• Eukaryotic systems for potential post-translational modifications:

  • Insect cells (Sf9, Sf21) with baculovirus expression systems

  • Mammalian cells (HEK293, CHO) for native-like processing

• Codon optimization: Important for expression in heterologous systems

• Construct design: Include appropriate fusion tags (His-tag has proven successful )

Researchers should perform small-scale expression trials to identify optimal conditions before scaling up, and consider testing both full-length protein and stable domains independently if initial expression attempts fail.

What purification strategies provide the highest purity C2orf74 for structural and functional studies?

The commercially available recombinant C2orf74 uses a C-terminal His tag and achieves ≥85% purity . Given its isoelectric point of 6.5 , researchers should consider:

• Primary purification: Immobilized metal affinity chromatography (IMAC)

  • Optimize imidazole concentrations for binding and elution

  • Test different metal ions (Ni²⁺, Co²⁺) for selective binding

• Secondary purification options:

  • Size exclusion chromatography to separate monomers from aggregates

  • Ion exchange chromatography (cation exchange at pH below 6.5)

• Buffer optimization:

  • Screen buffers around the isoelectric point (pH 6.0-7.0)

  • Test thermal stability using differential scanning fluorimetry

  • Evaluate additives (glycerol, salts, reducing agents) for stability

• Quality control:

  • SDS-PAGE for purity assessment

  • Western blot for identity confirmation

  • Mass spectrometry for accurate mass determination

  • Dynamic light scattering for homogeneity assessment

For challenging purifications, consider on-column refolding protocols or fusion with solubility-enhancing tags (MBP, SUMO) that can be removed after purification.

How can researchers verify proper folding and stability of recombinant C2orf74?

For an uncharacterized protein without established functional assays, folding assessment requires multiple complementary approaches:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to probe tertiary structure

  • Differential scanning calorimetry to determine thermal stability

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

• Stability assessment:

  • Thermal shift assays (Thermofluor) to identify stabilizing buffer conditions

  • Time-course analyses of aggregation propensity

  • Freeze-thaw stability tests for storage optimization

• Structural integrity:

  • Limited proteolysis followed by mass spectrometry to identify stable domains

  • Hydrogen-deuterium exchange mass spectrometry to probe solvent accessibility

  • Nuclear magnetic resonance (NMR) spectroscopy for structural assessment

The presence of two cysteine residues in the core sequence suggests monitoring disulfide bond formation may be important for verifying proper folding.

What systematic strategies can elucidate the function of an uncharacterized protein like C2orf74?

For functional characterization of C2orf74, researchers should implement a multi-dimensional approach:

• Localization studies:

  • Fluorescently tagged constructs for live-cell imaging

  • Subcellular fractionation followed by Western blotting

  • Immunofluorescence with organelle markers

• Interactome analysis:

  • Affinity purification coupled with mass spectrometry

  • Yeast two-hybrid screening

  • Proximity labeling methods (BioID, APEX)

• Loss-of-function studies:

  • CRISPR-Cas9 knockout in relevant cell lines

  • siRNA knockdown followed by phenotypic assays

  • Rescue experiments to confirm specificity

• Transcriptomic profiling:

  • RNA-seq after overexpression or knockdown

  • Pathway enrichment analysis of differentially expressed genes

• Disease-relevant assays based on known associations:

  • Autoimmune response measurements

  • Colon epithelial cell function

  • Assays related to Waardenburg syndrome phenotypes

The disease associations of C2orf74 with autoimmune disorders and colon diseases provide important context for designing targeted functional assays.

How should researchers approach C2orf74 expression profiling across tissues and disease states?

Understanding the expression pattern of C2orf74 is critical for functional insights:

The prognostic significance of C2orf74 expression in multiple cancer types suggests value in comparing expression levels between normal and malignant tissues, with potential clinical applications.

How can researchers investigate the link between C2orf74 and autoimmune disorders?

C2orf74 has been linked to autoimmune disorders, particularly ankylosing spondylitis . To investigate this connection:

• Genetic approaches:

  • SNP analysis in patient cohorts

  • Haplotype mapping in affected families

  • Assessment of linkage disequilibrium with known risk loci

• Expression analysis:

  • Quantify expression in immune cells from patients vs. controls

  • Compare expression in affected vs. unaffected tissues

  • Correlate expression with inflammatory markers

• Functional studies:

  • Examine impact on cytokine production in immune cells

  • Assess effects on NF-κB signaling

  • Evaluate influence on T cell activation and differentiation

• Animal models:

  • Evaluate C2orf74 expression in established autoimmune models

  • Generate transgenic models expressing disease-associated variants

  • Test therapeutic approaches targeting the protein or its pathway

The 50% identity between human and mouse orthologs indicates that mouse models may be suitable for mechanistic studies, though important functional differences could exist.

What methodological approaches can elucidate C2orf74's role in colorectal diseases?

Evidence suggests C2orf74 may play a role in colon diseases and colorectal cancer prognosis :

• Expression analysis in colorectal tissues:

  • Compare normal mucosa, adenomas, and carcinomas

  • Correlate with cancer stage and histological features

  • Assess relationship with microsatellite stability status

• Prognostic value assessment:

  • Kaplan-Meier survival analysis in patient cohorts

  • Multivariate analysis with established prognostic factors

  • Development of prognostic signatures including C2orf74

• Functional studies in colon cell models:

  • Establish knockdown/knockout in colorectal cancer cell lines

  • Assess impact on hallmarks of cancer (proliferation, migration, apoptosis)

  • Investigate effects on epithelial-to-mesenchymal transition

• Mechanistic investigations:

  • Identify signaling pathways affected by C2orf74 modulation

  • Examine relationship with known colorectal cancer drivers

  • Assess impact on inflammatory responses in the colon

What experimental designs can investigate the role of C2orf74 variants in Waardenburg syndrome?

A C2orf74 variant has been reported to segregate with Waardenburg syndrome 2 (WS2) in a large family, with some exceptions (individuals V:1 and V:2) . To investigate this association:

• Genetic approaches:

  • Sequence C2orf74 in additional WS2 families

  • Analyze co-occurrence with known WS2 genes (especially MITF)

  • Perform segregation analysis in extended families

• Functional characterization of variants:

  • Generate cellular models expressing the variant

  • Assess impact on melanocyte development and function

  • Test interaction with MITF, the primary gene associated with WS2

• Developmental studies:

  • Examine effects on neural crest migration

  • Assess impact on melanocyte differentiation

  • Investigate influence on cochlear development

• Mechanistic analysis:

  • Test whether C2orf74 variants affect MITF expression or activity

  • Assess impact on SOX10-MITF-TYR pathway

  • Determine if variants alter protein localization or stability

The co-occurrence of C2orf74 and MITF variants in WS2 patients suggests potential functional interaction that requires further investigation.

What controls and validation approaches are essential for C2orf74 research?

Given the uncharacterized nature of C2orf74, rigorous controls are critical:

• Protein expression controls:

  • Empty vector/tag-only controls

  • Irrelevant protein expressed with the same system

  • Multiple expression systems to confirm findings

• Knockdown/knockout controls:

  • Non-targeting siRNA/sgRNA

  • Rescue experiments with wild-type protein

  • Phenotype comparison with known genes in related pathways

• Interaction study controls:

  • Tag-only pull-downs

  • Competition with untagged protein

  • Reciprocal co-immunoprecipitation

• Antibody validation:

  • Peptide competition assays

  • Testing in knockout/knockdown samples

  • Cross-reactivity assessment with related proteins

• Expression analysis controls:

  • Multiple reference genes for qPCR

  • Comparison across detection methods (RNA-seq, qPCR, protein)

  • Cell-type specific markers in heterogeneous samples

For disease studies, using multiple patient cohorts and replication in independent populations is essential to confirm associations with confidence.

How should researchers select appropriate cell models for C2orf74 functional studies?

Cell model selection should be guided by expression patterns and disease associations:

• Based on disease associations:

  • Immune cells (T cells, B cells) for autoimmune studies

  • Colorectal cell lines (Caco-2, HT-29) for colon disease research

  • Melanocytes for Waardenburg syndrome studies

• Expression-guided selection:

  • Analyze public databases to identify high-expressing cell types

  • Consider developmental context for embryonic studies

  • Match model to relevant tissue/organ system

• Technical considerations:

  • Transfection efficiency for overexpression studies

  • Genetic manipulation capability (CRISPR amenability)

  • Availability of assay systems for functional readouts

• Physiological relevance:

  • Primary cells vs. cell lines

  • 3D culture systems (organoids, spheroids)

  • Co-culture models to study cell-cell interactions

The prognostic significance of C2orf74 in multiple cancer types suggests cancer cell lines may provide valuable insights, particularly colorectal cancer models where expression correlates with survival outcomes.

What strategies can overcome challenges in studying uncharacterized proteins like C2orf74?

Researching uncharacterized proteins presents unique challenges requiring specialized approaches:

• Knowledge gap strategies:

  • Start with subcellular localization to narrow potential functions

  • Use comparative genomics with better-characterized orthologs

  • Apply domain prediction to suggest functional categories

• Technical approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • High-content screening to identify phenotypic changes

  • Network analysis to place the protein in functional context

• Collaboration strategies:

  • Form consortia with complementary expertise

  • Establish resource sharing for reagents and models

  • Create centralized databases for findings

• Funding considerations:

  • Target special initiatives for uncharacterized proteins

  • Emphasize disease relevance in proposals

  • Frame research questions within broader biological contexts

For C2orf74 specifically, leveraging its disease associations with autoimmune disorders , colon diseases , and Waardenburg syndrome provides entry points for functional investigation.

How might multi-omics approaches advance understanding of C2orf74?

Integrative approaches combining multiple data types can accelerate functional discovery:

• Data integration strategies:

  • Correlate transcriptomics with proteomics after C2orf74 modulation

  • Link genomic variants to expression patterns (eQTL analysis)

  • Integrate epigenomic data to understand regulation

  • Apply metabolomics to identify affected pathways

• Network biology approaches:

  • Construct protein-protein interaction networks

  • Develop gene regulatory networks

  • Identify signaling pathway impacts

• Computational methods:

  • Apply machine learning to identify patterns across datasets

  • Use systems biology modeling to predict functional impacts

  • Develop causal inference methods to establish directionality

• Public data mining:

  • Leverage TCGA/GTEx data for expression patterns

  • Analyze GWAS data for disease associations

  • Mine proteomics databases for post-translational modifications

The prognostic significance of C2orf74 in multiple cancer types provides a valuable context for multi-omics investigation, particularly regarding its potential role in cancer progression mechanisms.

What emerging technologies could accelerate functional characterization of C2orf74?

Several cutting-edge technologies offer promising approaches for uncharacterized proteins:

• Advanced imaging:

  • Super-resolution microscopy for detailed localization

  • Live-cell imaging with optogenetic control

  • Correlative light and electron microscopy

• Genome engineering:

  • CRISPR-based screening (knockout, activation, interference)

  • Base editing for precise mutation introduction

  • CRISPR-Cas13 for RNA-level manipulation

• Single-cell technologies:

  • Multimodal single-cell analysis (RNA, protein, chromatin)

  • Spatial transcriptomics for tissue context

  • Single-cell proteomics for protein-level insights

• Structural biology innovations:

  • Cryo-electron microscopy for structure determination

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Integrative structural biology combining multiple data types

For C2orf74 specifically, applying these technologies in the context of its disease associations could yield particularly valuable insights into its function and potential as a therapeutic target.

How can researchers develop C2orf74-targeted therapeutics based on current knowledge?

Despite limited functional understanding, several approaches can guide therapeutic development:

• Target validation strategies:

  • Confirm disease relevance through genetic models

  • Identify critical interaction partners as potential co-targets

  • Establish biomarkers for patient stratification

• Therapeutic modalities to consider:

  • Small molecule inhibitors targeting protein interactions

  • Biologics (antibodies, peptides) for specific binding

  • RNA-based therapies (antisense, siRNA) for expression modulation

• Screening approaches:

  • Structure-based virtual screening if models available

  • Phenotypic screening in disease-relevant assays

  • Fragment-based drug discovery

• Translational considerations:

  • Develop appropriate animal models for efficacy testing

  • Establish patient-derived systems for personalized approaches

  • Identify potential combination therapies based on pathway analysis

The association of C2orf74 with multiple diseases provides several potential therapeutic directions, with autoimmune applications perhaps being most immediately promising given the strong link to ankylosing spondylitis.