Recombinant Mouse Uncharacterized protein C2orf82 homolog

What is the mouse C2orf82 homolog protein?

The mouse C2orf82 homolog (UniProt ID: Q9CXL7) is a protein encoded by a gene homologous to the human C2orf82 gene (chromosome 2 open reading frame 82). It is also referred to as "secondary ossification center associated regulator of chondrocyte maturation" in some databases. The protein consists of 121 amino acids with the expression region spanning from amino acids 25-121. The full amino acid sequence is: AEGPQEPDPTLWNEPIELPSGEGPLESTSHNQEFAVSGPPFPTSAPAPEDSTPPARVDQDGGSLGPGAIAAIVIAALLATCVVLALVVVALRKFSAS . This protein has a transmembrane domain, suggesting it may function as a membrane-associated protein.

What is currently known about the biological function of mouse C2orf82 homolog?

Current research suggests that the C2orf82 homolog may play a role in neurodevelopmental processes. Epigenetic studies have identified that this gene undergoes differential methylation that correlates with expression levels. Specifically, ADHD risk alleles correlate with increased methylation and decreased expression of C2orf82 . Genetic variants in C2orf82 have been correlated with variations in brain volumes, particularly in the accumbens and caudate regions, suggesting potential roles in brain development or function . Additionally, its classification as a "secondary ossification center associated regulator of chondrocyte maturation" hints at possible roles in bone development processes .

How is the recombinant mouse C2orf82 homolog protein typically produced?

The recombinant mouse C2orf82 homolog is typically produced through standard recombinant protein expression techniques. This involves:

• Gene cloning: The C2orf82 gene sequence is isolated and inserted into appropriate expression vectors (plasmids) .

• Transformation: The recombinant plasmid is introduced into an expression system, which could be bacterial (e.g., E. coli BL21), yeast, or mammalian cells .

• Protein expression: The host cells are cultured under conditions that induce expression of the target protein .

• Purification: The expressed protein is purified, typically using affinity chromatography methods such as nickel-NTA affinity chromatography if a His-tag is incorporated into the recombinant protein design .

The purified recombinant protein is then stored in appropriate buffer conditions, often with 50% glycerol in a Tris-based buffer at -20°C or -80°C for extended storage .

What expression systems are most effective for producing recombinant mouse C2orf82 homolog?

When selecting an expression system for the mouse C2orf82 homolog, researchers should consider several factors:

E. coli expression system:

• Advantages: Simple, cost-effective, high protein yield

• Limitations: Lacks post-translational modifications; may form inclusion bodies if the protein contains transmembrane domains (as C2orf82 does)

Mammalian expression systems:

• Advantages: Proper folding and post-translational modifications; better for membrane proteins

• Limitations: More expensive, lower yield, longer production time

Based on the protein characteristics (containing a transmembrane domain), a mammalian expression system such as HEK293 or CHO cells might be more appropriate for functional studies, while E. coli might be sufficient for structural or antibody production purposes . The choice of expression tags (His-tag, GST, etc.) should be determined during the production process based on the specific experimental requirements .

What are the optimal storage conditions for recombinant mouse C2orf82 homolog?

For optimal stability and activity of recombinant mouse C2orf82 homolog:

• Store the purified protein at -20°C for routine use, or at -80°C for extended storage periods

• Use a Tris-based buffer with 50% glycerol, optimized for this specific protein

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity

• Consider storing working aliquots at 4°C for up to one week to minimize freeze-thaw cycles

• For long-term experiments, create multiple small aliquots during initial purification

These conditions help maintain protein stability and functional integrity for experimental applications.

What purification strategies yield the highest purity recombinant mouse C2orf82 homolog?

A multi-step purification strategy is recommended for obtaining high-purity recombinant mouse C2orf82 homolog:

• Affinity chromatography: If the recombinant protein contains a His-tag, nickel-NTA affinity chromatography is the preferred first step. Elution with 500 mM imidazole typically yields good initial purification .

• Size exclusion chromatography (SEC): This can separate the target protein from aggregates and smaller contaminants based on molecular size.

• Ion exchange chromatography: Depending on the theoretical isoelectric point of the mouse C2orf82 homolog, either cation or anion exchange chromatography can be employed for further purification.

• Quality control: Assess protein purity using SDS-PAGE and Western blot analysis with anti-His antibodies or specific antibodies against the C2orf82 protein .

The purification protocol should be optimized based on the specific expression system used and the experimental requirements for protein purity.

How does methylation affect C2orf82 expression and what methodologies are best for studying this relationship?

Research has identified that C2orf82 undergoes allele-specific methylation (ASM) that correlates with its expression levels. ADHD risk alleles correlate with increased methylation and decreased expression of C2orf82 . To study this relationship:

• Bisulfite sequencing: For quantitative analysis of DNA methylation at specific CpG sites in the C2orf82 promoter region.

• Methylation-specific PCR (MSP): To detect the presence of methylation in specific regions.

• Chromatin immunoprecipitation (ChIP): To identify proteins associated with methylated regions of C2orf82.

• Expression analysis: Using RT-qPCR, RNA-seq, or protein quantification methods to correlate methylation status with expression levels.

• CRISPR-based epigenetic editing: To artificially manipulate methylation status and observe effects on expression.

These methodologies can help elucidate the complex relationship between genetic variation, methylation, and gene expression in the context of neurodevelopmental disorders.

What are the current challenges in studying C2orf82 function in mouse models?

Several significant challenges exist in studying C2orf82 function:

• Limited functional characterization: As an "uncharacterized protein," the precise cellular function remains unclear, making it difficult to design targeted functional assays.

• Protein localization: The presence of a transmembrane domain suggests membrane localization, but experimental verification of subcellular localization is essential for functional studies.

• Knockout/knockdown models: Generating and validating specific knockout or knockdown models can be challenging without knowing the protein's function or reliable antibodies.

• Developmental timing: If C2orf82 functions in neurodevelopment, timing of expression during development becomes critical for experimental design.

• Translational relevance: Establishing the relevance of mouse findings to human neurodevelopmental disorders requires careful validation across species.

Addressing these challenges requires multidisciplinary approaches combining genomics, proteomics, and developmental biology techniques.

How can researchers effectively study the relationship between C2orf82 genetic variants and brain volume changes?

To investigate the relationship between C2orf82 genetic variants and brain volume:

• Genetically modified mouse models: Generate mice with specific C2orf82 variants corresponding to human variants associated with brain volume changes.

• Neuroimaging techniques: Employ micro-MRI or micro-CT scanning to quantify brain structures, focusing particularly on accumbens and caudate volumes that have shown correlation with C2orf82 variants .

• Integration with human data: Compare findings with human neuroimaging studies from resources like ENIGMA consortium data.

• Histological analysis: Complement imaging with detailed histological examination of relevant brain regions.

• Developmental timeline: Examine brain development across multiple timepoints to identify when volume differences first appear.

This comprehensive approach can help establish causality between genetic variants, gene expression changes, and anatomical differences in brain structure.

What controls should be included when studying recombinant mouse C2orf82 homolog in immunization experiments?

When designing immunization experiments involving recombinant C2orf82 homolog, comprehensive controls are essential:

Additionally, time-course sampling and dose-response relationships should be established to fully characterize immune responses. The experimental design should include appropriate sample sizes for statistical power and consider both cellular and humoral immune responses .

How can researchers address the issue of protein solubility when working with recombinant mouse C2orf82 homolog?

The presence of a transmembrane domain in C2orf82 homolog can present solubility challenges during recombinant expression and purification. Consider these approaches:

• Solubility tags: Fusion with solubility-enhancing tags like GST, MBP, or SUMO can improve solubility during expression.

• Detergent screening: Systematic testing of different detergents (non-ionic, zwitterionic, etc.) to identify optimal solubilization conditions.

• Truncation constructs: Express soluble domains separately by removing the transmembrane region for certain applications.

• Co-expression with chaperones: Express with molecular chaperones to improve folding and solubility.

• Buffer optimization: Systematically test various pH conditions, salt concentrations, and additives to identify optimal solubilization conditions.

• Carrier proteins: Consider using carrier proteins such as bovine serum albumin (BSA) to enhance stability, though for some applications carrier-free versions may be preferred .

Appropriate reconstitution protocols after lyophilization are also critical, typically using sterile PBS with or without carrier proteins depending on the application .

What bioassays can effectively measure the functional activity of recombinant mouse C2orf82 homolog?

Due to the limited functional characterization of C2orf82, developing activity assays requires an investigative approach:

• Cell-based proliferation/differentiation assays: Based on its potential role in chondrocyte maturation, assess effects on relevant cell lines (chondrocytes, osteoblasts) using proliferation or differentiation markers.

• Brain-derived cell models: Given the association with brain volumes, test effects on neuronal cell lines or primary neuronal cultures, measuring parameters such as neurite outgrowth, synapse formation, or calcium signaling.

• Binding assays: Develop binding assays to identify potential protein-protein interactions using techniques such as co-immunoprecipitation or surface plasmon resonance.

• Reporter gene assays: Design reporter constructs to measure potential transcriptional effects if C2orf82 influences gene expression.

• Methylation analysis: Given the correlation between C2orf82 variants and differential methylation, develop assays to measure changes in DNA methylation patterns in relevant genomic regions.

Since C2orf82 function is not fully characterized, researchers should consider multiple assays and validate findings across different experimental systems.

How should researchers interpret contradictory findings between C2orf82 methylation status and expression levels?

When faced with contradictory findings regarding C2orf82 methylation and expression:

• Context-specific regulation: Consider that the relationship between methylation and expression may be context-dependent, varying across:

  • Different brain regions

  • Developmental stages

  • Cell types

  • Environmental conditions

• Technical considerations:

  • Assess whether contradictions arise from differences in methylation analysis techniques

  • Evaluate expression measurement methodologies (RNA-seq, qPCR, protein levels)

  • Consider sample preparation differences

• Genetic background effects:

  • Analyze whether specific genetic variants modify the methylation-expression relationship

  • Examine potential effects of distant regulatory elements

• Statistical approach:

  • Perform meta-analysis when multiple studies exist

  • Use multivariate analyses to identify confounding variables

  • Apply causal inference methods to distinguish correlation from causation

• Biological validation:

  • Use CRISPR-based approaches to directly manipulate methylation and measure expression

  • Employ reporter assays with methylated vs. unmethylated promoters

The research findings from studies on ADHD risk alleles suggest a correlation between increased methylation and decreased expression of C2orf82 , but this pattern may not be universal across all experimental conditions or tissues.

What bioinformatic approaches are most appropriate for analyzing C2orf82 structural and functional characteristics?

A comprehensive bioinformatic analysis of C2orf82 should include:

• Structural prediction:

  • Secondary structure prediction using tools like PSIPRED

  • Transmembrane domain prediction using TMHMM or Phobius

  • 3D structure prediction using AlphaFold2 or RoseTTAFold

  • Post-translational modification site prediction

• Functional annotation:

  • Gene Ontology (GO) term analysis

  • Protein family classification

  • Conserved domain identification

  • Motif analysis for functional sites

• Evolutionary analysis:

  • Phylogenetic profiling across species

  • Selection pressure analysis (dN/dS ratios)

  • Identification of conserved regions that may indicate functional importance

• Interaction networks:

  • Text-mining for potential interactors

  • Co-expression analysis across tissues

  • Protein-protein interaction prediction

• Integration with genetic data:

  • Analysis of SNPs and their potential functional effects

  • Linkage disequilibrium patterns

  • eQTL analysis to correlate genetic variants with expression levels

These approaches can provide valuable insights into the potential functions of this relatively uncharacterized protein and guide experimental design.

How can researchers effectively combine mouse and human data to understand C2orf82 function in neurodevelopmental disorders?

Translating findings between mouse C2orf82 homolog and human C2orf82 requires careful integration:

• Sequence and structural homology:

  • Perform detailed sequence alignment to identify conserved domains

  • Compare protein structure predictions between species

  • Map human disease-associated variants onto mouse protein sequence

• Expression pattern comparison:

  • Compare spatial and temporal expression patterns during development

  • Analyze expression correlation with other genes across species

  • Examine cell-type specific expression in equivalent tissues

• Functional equivalence testing:

  • Use cross-species rescue experiments (human gene in mouse knockout)

  • Compare phenotypes of mouse models with human disorder characteristics

  • Test whether human variants produce similar effects when introduced to mouse models

• Multi-omics integration:

  • Integrate transcriptomic, proteomic, and epigenomic data across species

  • Use systems biology approaches to identify conserved networks

  • Employ machine learning to identify patterns across datasets

• Translational validation:

  • Design experiments that can be replicated in both human and mouse systems

  • Utilize patient-derived cells alongside mouse models

  • Validate mouse findings using human post-mortem tissue or neuroimaging data

This integrative approach can identify which aspects of C2orf82 function are conserved across species and most relevant to human neurodevelopmental disorders like ADHD, where C2orf82 variants have been implicated .