Recombinant Human Uncharacterized protein C3orf43 (C3orf43)

What is C3orf43 and why is it classified as an "uncharacterized protein"?

C3orf43 (Chromosome 3 Open Reading Frame 43) is a 214-amino acid human protein encoded by a gene located on chromosome 3. It is classified as "uncharacterized" because its biological function has not been fully determined through experimental validation. According to current proteomic databases, C3orf43 belongs to the substantial portion of the human proteome whose functions remain to be discovered . The protein contains the amino acid sequence MNNETTTLISLKEAMKRVDHKLQALETQFKELDFTKDNLMQKFEHHSKALASQAAQDEMWTAVRALQLTSMELNILYSYVIEVLICLHTRVLEKLPDLVRGLPTLASVLRRKVKNKRRVVWESILEECGLQEGDITALCTFFIARGNKAEHYTAKVRQMYIRDVTFLITNMVKNQALQDSLLRAVQVIEKGKAVRTPEKQKSSLEELIPSVKN . Despite being uncharacterized, addressing such proteins is crucial as they may represent undiscovered biological functions or regulatory mechanisms.

What expression systems are most effective for producing recombinant C3orf43?

E. coli is the currently documented expression system for producing recombinant C3orf43 with an N-terminal His tag . While E. coli offers advantages for protein expression including rapid growth, high yields, and cost-effectiveness, researchers should consider alternative expression systems when studying potential post-translational modifications or when protein solubility is a concern. Potential alternative expression systems include:

The choice should be guided by research objectives, particularly whether native post-translational modifications are essential for the study.

What is the recommended protocol for reconstitution and storage of recombinant C3orf43?

For optimal stability and activity, recombinant C3orf43 requires specific handling procedures. The recommended protocol based on available data includes:

• Centrifuge the vial briefly before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is standard/recommended)

• Aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles

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

The protein is supplied as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . These storage conditions are designed to maintain protein stability and prevent degradation, which is particularly important for uncharacterized proteins where functional assays may not be immediately available to verify activity.

What bioinformatic approaches can help predict C3orf43 function?

When working with uncharacterized proteins like C3orf43, bioinformatic analysis provides valuable insights into potential functions. A multi-layered approach is recommended:

Recent advances in AI-based structure prediction tools like AlphaFold2 can provide three-dimensional structural models of C3orf43, potentially revealing functional sites not apparent from sequence analysis alone. This integrative approach has proven effective for initial characterization of proteins similar to C3orf43 in recent studies of the human proteome .

What experimental approaches are most effective for functional characterization of C3orf43?

The functional characterization of uncharacterized proteins like C3orf43 requires a systematic multi-omics approach:

• Interactome Analysis: Employ techniques such as affinity purification-mass spectrometry (AP-MS), BioID, or APEX proximity labeling to identify protein interaction partners of C3orf43. Recent studies have demonstrated that characterizing the interactome of previously uncharacterized proteins can provide significant insights into their cellular roles .

• Transcriptome Analysis after Genetic Manipulation: CRISPR-Cas9 knockout or siRNA knockdown of C3orf43 followed by RNA-seq analysis can reveal affected pathways. Similar approaches with other uncharacterized proteins have identified unexpected roles in cellular processes .

• Cellular Phenotyping: High-content imaging after C3orf43 manipulation can reveal morphological changes or alterations in cellular processes. This approach has been particularly useful in characterizing proteins with roles in cell division, migration, or organelle homeostasis.

• Micronutrient Response Analysis: Based on findings with other uncharacterized human proteins, testing expression responses to essential micronutrients like manganese and selenium might reveal regulatory connections, as demonstrated for C9orf85 and CXorf38 .

• Translatome Sequencing: This approach can identify potential alternative splicing isoforms of C3orf43, similar to methods used to identify 50 novel protein isoforms in hepatocellular carcinoma studies .

These complementary approaches provide a comprehensive strategy to elucidate C3orf43 function from multiple perspectives.

How can researchers design statistically robust experiments to detect phenotypic effects of C3orf43 manipulation?

Designing statistically robust experiments for uncharacterized proteins presents unique challenges due to unknown effect sizes and potential experimental noise. A well-designed experimental strategy should incorporate:

• Power Analysis for Panel Data: When collecting time-series data after C3orf43 manipulation, researchers should account for non-i.i.d. error structures. Traditional methods for experimental design often yield incorrectly powered experiments with proper inference when errors are correlated .

• Sample Size Determination:

• Control for Multiple Testing: When screening for phenotypic effects across multiple cellular pathways, implement appropriate multiple testing corrections such as Benjamini-Hochberg procedure or false discovery rate control.

• Heterogeneous Treatment Effects: Consider potential variation in C3orf43 effects across different cell types or genetic backgrounds, which may require stratified analysis or interaction terms in statistical models .

Monte Carlo simulations based on preliminary data can help determine appropriate sample sizes and experimental designs, particularly when serial correlation is expected in longitudinal measurements after C3orf43 manipulation .

What approaches can determine if C3orf43 undergoes post-translational modifications?

Post-translational modifications (PTMs) often regulate protein function and can be especially important for understanding uncharacterized proteins. A comprehensive strategy to identify PTMs on C3orf43 includes:

• Predictive Analysis: Use computational tools like NetPhos, UbPred, and GPS to predict potential phosphorylation, ubiquitination, and other modification sites based on the C3orf43 sequence.

• Mass Spectrometry-Based Detection:

  • Employ enrichment strategies specific to different PTMs (phosphopeptide enrichment, ubiquitin remnant profiling)

  • Analyze C3orf43 expressed in mammalian systems rather than the E. coli system currently documented

  • Use multiple proteases to maximize sequence coverage

  • Consider targeted MS approaches once candidate sites are identified

• PTM-Specific Antibody Detection: Develop or utilize antibodies against common PTMs to probe whether C3orf43 undergoes modifications in different cellular contexts.

• Writer/Eraser Enzyme Screening: Test whether known modification enzymes can act on C3orf43 in vitro, similar to studies on SETD7, which has been shown to methylate over 30 non-histone substrates .

• Functional Impact Analysis: Create site-directed mutants of predicted PTM sites to assess their impact on C3orf43 localization, interactions, or cellular phenotypes when identified.

This systematic approach can help uncover regulatory mechanisms controlling C3orf43 function and potentially place it within known signaling networks.

How can translatome sequencing help characterize potential isoforms of C3orf43?

Translatome sequencing offers powerful advantages for identifying and characterizing protein isoforms of uncharacterized genes like C3orf43:

• Identification of Novel Isoforms: Translatome sequencing can detect ribosome-associated mRNAs, revealing actively translated transcripts including previously unidentified splice variants. Research has shown this approach successfully identified 50 novel protein isoforms in a hepatocellular carcinoma cell line .

• Quantification of Isoform-Specific Translation Efficiency: This approach can determine which C3orf43 transcript variants are preferentially translated under different conditions, providing insights into potential isoform-specific functions.

• Tissue-Specific Translation Patterns: By comparing translatome data across tissues, researchers can identify cell type-specific expression patterns of C3orf43 isoforms that may correlate with specialized functions.

• Integration with Proteomics:

This multi-layered approach has been successfully applied to identify novel isoforms of other uncharacterized proteins and can expand our understanding of C3orf43's potential functional diversity .

What disease contexts might be relevant for investigating C3orf43 function?

While direct evidence linking C3orf43 to specific diseases is currently limited, several research approaches can help identify potential disease relevance:

By investigating C3orf43 through these complementary approaches, researchers may uncover unexpected connections to human pathologies, potentially leading to new therapeutic targets or diagnostic markers.