Recombinant Uncharacterized protein C34C12.7 (C34C12.7)

What is C34C12.7 protein and what organism is it found in?

C34C12.7 is an uncharacterized protein found in the nematode Caenorhabditis elegans (C. elegans), a model organism widely used in biological research. The protein contains a PAN domain, which confers protein-protein or protein-carbohydrate interaction capabilities, suggesting potential roles in molecular recognition processes . Current research indicates this protein may be membrane-associated and potentially GPI-anchored based on experimental evidence from studies using phosphatidylinositol-specific phospholipase C (PI-PLC) treatment . While the protein remains functionally uncharacterized, its conservation across nematode species suggests biological significance.

What is known about the structure and domains of C34C12.7?

The protein C34C12.7 contains a PAN domain, which is known to mediate protein-protein or protein-carbohydrate interactions . The expression region of C34C12.7 spans amino acids 21-284 of the full-length protein . Structurally, based on experimental approaches used in research, C34C12.7 appears to be membrane-associated and may be GPI-anchored, which would localize it to the cell surface . The presence of the PAN domain suggests potential involvement in cell adhesion, receptor-ligand interactions, or other molecular recognition processes. Further structural characterization through techniques such as X-ray crystallography or cryo-electron microscopy would be valuable for elucidating its three-dimensional structure and functional interfaces.

What are the known orthologs of C34C12.7 in other species?

Bm2565 has been identified as an ortholog of C. elegans C34C12.7 in Brugia malayi, a parasitic nematode that causes lymphatic filariasis . This orthology suggests evolutionary conservation of this protein across nematode species, which may indicate important biological functions. The conservation of the PAN domain between these orthologs further supports the functional significance of this protein in nematode biology . Researchers studying C34C12.7 should consider comparative analyses with Bm2565 to gain insights into conserved functions. The study of both proteins in parallel could provide valuable information about the functional evolution of PAN domain-containing proteins across nematode species and potentially reveal species-specific adaptations in their molecular mechanisms.

What experimental approaches are recommended for studying C34C12.7?

Multiple complementary approaches are recommended for studying the uncharacterized protein C34C12.7. Membrane fractionation through ultracentrifugation at 100,000 x g has proven effective for isolating membrane-associated fractions containing this protein . Treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) can be employed to release GPI-anchored proteins, followed by protein identification using mass spectrometry (LC-MS/MS) . For in vivo functional studies, RNAi knockdown experiments in C. elegans provide valuable insights, though researchers should be cautious as RNAi may affect multiple family members leading to more severe phenotypes than specific mutations .

When conducting membrane isolation, researchers should implement a protocol similar to the following:

• Homogenize tissue in cold sucrose buffer with protease inhibitors

• Centrifuge at 1000 x g to remove debris

• Ultracentrifuge supernatant at 100,000 x g to pellet membrane fraction

• Wash membrane pellet with 150 mM Tris-HCl, pH 8.0

• Resuspend in PBS and treat with PI-PLC if investigating GPI-anchoring

For detection of the protein, fluorescent aerolysin (FLAER) binding assays have been used successfully to visualize GPI-anchored proteins on nitrocellulose membranes .

How can recombinant C34C12.7 be effectively expressed and purified?

Effective expression and purification of recombinant C34C12.7 requires careful consideration of expression systems and buffer conditions. Based on available information, the recombinant protein can be stored in a Tris-based buffer with 50% glycerol to maintain stability . For expression, researchers should consider using eukaryotic expression systems such as insect cells or yeast rather than bacterial systems, as these may better accommodate potential post-translational modifications including GPI-anchoring . The expression construct should include the region spanning amino acids 21-284, which encompasses the functional domains of the protein .

For purification, affinity tags such as His-tag or GST-tag can be incorporated into the expression construct, followed by appropriate affinity chromatography. Given the membrane association of the native protein, detergents may be necessary during purification to maintain solubility. After initial purification, size exclusion chromatography can be employed to achieve higher purity. For storage, a buffer containing 50% glycerol appears suitable, and aliquots should be stored at -20°C or -80°C for extended storage to prevent repeated freeze-thaw cycles which may compromise protein integrity .

What methods are suitable for investigating protein interactions of C34C12.7?

Given that C34C12.7 contains a PAN domain known for mediating protein-protein or protein-carbohydrate interactions, several analytical methods are suitable for investigating its interactome. Co-immunoprecipitation using antibodies against C34C12.7 or potential binding partners can identify protein complexes in vivo . Yeast two-hybrid screening provides an unbiased approach to identify novel interaction partners, especially when using the PAN domain as bait. For more quantitative measurements, surface plasmon resonance or biolayer interferometry can determine binding kinetics and affinities.

Pull-down assays using tagged recombinant C34C12.7 followed by mass spectrometry represent a powerful approach for identifying binding partners in complex lysates. For structural characterization of interactions, X-ray crystallography or cryo-electron microscopy of protein complexes can reveal molecular details of binding interfaces. Proximity-based labeling techniques such as BioID or APEX can identify proteins that are in close proximity to C34C12.7 in vivo, potentially revealing functional interaction networks. When investigating carbohydrate interactions, glycan arrays can be employed to identify specific carbohydrate structures recognized by the PAN domain .

How might C34C12.7 relate to the Sma/Mab signaling pathway in C. elegans?

The potential relationship between C34C12.7 and the Sma/Mab signaling pathway presents an intriguing avenue for research. The Sma/Mab pathway is a TGF-β-like signaling cascade that regulates body size and male tail development in C. elegans . To investigate C34C12.7's role in this pathway, researchers should employ genetic approaches including generation of C34C12.7 mutant strains and analysis of their phenotypes in comparison to known Sma/Mab pathway mutants. Epistasis analysis, placing C34C12.7 mutants in the background of mutations in core Sma/Mab pathway components, can help determine where in the pathway C34C12.7 functions, if at all.

Biochemical approaches should include testing for physical interactions between C34C12.7 and known Sma/Mab pathway proteins such as SMA-2, SMA-3, and SMA-4 (Smad proteins) or the receptors SMA-6 and DAF-4 . Given that protein phosphatases have been implicated in modulating the Sma/Mab pathway, and research has been conducted on identifying such phosphatases, investigating whether C34C12.7 has any influence on phosphorylation states of pathway components would be valuable . Reporter assays monitoring Sma/Mab target gene expression in the presence and absence of functional C34C12.7 could provide functional evidence for its involvement in the pathway.

What are the key challenges in working with uncharacterized proteins like C34C12.7?

Working with uncharacterized proteins like C34C12.7 presents several significant challenges for researchers. First, the lack of established functional assays makes it difficult to confirm that purified recombinant protein retains its biological activity, necessitating the development of novel assays based on predicted functions from domain analysis . Second, without detailed structural information, designing expression constructs becomes more challenging, potentially leading to issues with protein folding, solubility, or stability during purification attempts. Third, the absence of specific antibodies against uncharacterized proteins often requires researchers to rely on epitope tagging, which may interfere with native protein function or localization.

Additionally, interpreting phenotypes from genetic manipulations (knockouts, RNAi) becomes more complex without a framework of established functions to compare against . The potential for redundancy with related proteins further complicates the analysis of loss-of-function experiments. Researchers must employ multiple complementary approaches (biochemical, genetic, structural, computational) to gradually build evidence for protein function, which demands significant time and resource investment. Finally, publication of results can be challenging when working with uncharacterized proteins, as reviewers often expect comprehensive functional characterization rather than incremental advances in understanding.

How should researchers approach contradictory data about C34C12.7's function?

When confronted with contradictory data regarding C34C12.7's function, researchers should implement a systematic approach to resolve discrepancies. First, all experimental conditions should be meticulously reviewed to identify potential variables that might explain the contradictions, including differences in protein preparation, experimental systems, or detection methods . Second, independent validation using orthogonal techniques should be performed to confirm observations. For instance, if protein interaction studies suggest one function while genetic studies suggest another, researchers should employ a third approach such as in vivo imaging or biochemical assays.

Cross-laboratory validation can be particularly valuable, as it eliminates lab-specific biases or technical variations. Researchers should consider that contradictory data may reflect true biological complexity, such as context-dependent protein functions or tissue-specific roles. Careful documentation of all experimental parameters is essential for identifying sources of variation . When publishing results with contradictory findings, researchers should transparently present all data, including contradictions, and discuss potential explanations for the discrepancies. Finally, computational approaches, including molecular dynamics simulations or evolutionary analyses comparing C34C12.7 with its orthologs like Bm2565, may provide additional insights to help resolve contradictions .

What are best practices for record-keeping when researching novel proteins like C34C12.7?

Effective record-keeping is especially crucial when researching novel proteins like C34C12.7, as inconsistent documentation can hinder progress and lead to irreproducible results. According to established best practices, researchers should maintain detailed laboratory notebooks documenting all experimental procedures, conditions, and observations . For recombinant protein work, comprehensive records should include expression construct designs, expression conditions, purification protocols, buffer compositions, and storage conditions. All raw data, including unsuccessful experiments, should be preserved to avoid repeating unproductive approaches .

Records should be maintained at three distinct levels: individual researcher notes, research group standardized protocols, and departmental/institutional archives . Digital data should be backed up regularly with clear file naming conventions that include dates, experiment codes, and brief descriptions. For collaborative projects involving C34C12.7, a centralized data repository with standardized formats ensures consistency across research teams. Sample tracking systems should document the preparation, storage location, and usage history of all protein preparations. Regular research group meetings to discuss methodological challenges and successes can enhance collective knowledge and improve record-keeping practices . These comprehensive record-keeping practices are essential for ensuring experimental reproducibility and facilitating the eventual functional characterization of C34C12.7.

What control experiments are essential when studying C34C12.7?

When studying the uncharacterized protein C34C12.7, several essential control experiments must be incorporated to ensure reliable and interpretable results. For protein expression and purification studies, positive controls using well-characterized proteins with similar properties (e.g., other PAN domain-containing proteins) should be included to validate experimental conditions . Negative controls, such as mock purifications from cells not expressing C34C12.7, are crucial for distinguishing specific signals from background. When investigating membrane association or GPI-anchoring, researchers should include known GPI-anchored proteins as positive controls and cytosolic proteins as negative controls in PI-PLC treatment experiments .

For functional studies using RNAi or CRISPR-based approaches, controls targeting genes with well-established phenotypes should be included to confirm the efficacy of the experimental system . When studying protein-protein interactions, several controls are essential: (1) a non-specific protein unlikely to interact with C34C12.7, (2) validation using reciprocal co-immunoprecipitation, and (3) competition assays with untagged protein to confirm specificity. Domain function can be validated through mutagenesis of key residues, with wild-type protein serving as a positive control. For all experiments, technical replicates (testing the same sample multiple times) and biological replicates (using independently prepared samples) are necessary to ensure reproducibility and statistical validity of findings.

How can predicted functional domains in C34C12.7 be experimentally validated?

Experimental validation of the predicted PAN domain and other functional elements in C34C12.7 requires a multi-faceted approach. Site-directed mutagenesis of conserved residues within the PAN domain followed by functional assays represents a primary approach to validate domain functionality . By comparing wild-type protein with mutant variants in binding assays, researchers can determine which residues are critical for domain function. Domain swapping experiments, where the PAN domain of C34C12.7 is replaced with analogous domains from well-characterized proteins, can test for functional complementation and domain autonomy.

Structural studies using X-ray crystallography, nuclear magnetic resonance (NMR), or cryo-electron microscopy can confirm the predicted three-dimensional structure of the domain . For validating protein-protein interactions mediated by the PAN domain, direct binding assays such as surface plasmon resonance with purified interaction partners provide quantitative measurements of binding affinities and kinetics. In vivo approaches include creating transgenic C. elegans expressing domain-deletion variants of C34C12.7 and assessing rescue of mutant phenotypes. Comparative studies with known PAN domain-containing proteins can provide functional insights through sequence and structural homology. Finally, evolutionary conservation analysis across species can identify the most critical residues within the domain that have been maintained through selective pressure .

What are the potential biological functions of C34C12.7 based on current evidence?

Based on current evidence, several potential biological functions can be hypothesized for C34C12.7. The presence of a PAN domain suggests a role in protein-protein or protein-carbohydrate interactions, potentially in cell adhesion, receptor-ligand binding, or extracellular matrix interactions . Its apparent membrane association and potential GPI-anchoring indicate a cell surface localization, consistent with roles in cell signaling, environmental sensing, or intercellular communication . The orthology relationship with Bm2565 in Brugia malayi suggests an evolutionarily conserved function across nematode species, pointing to a fundamental biological role rather than a species-specific adaptation .

The possible connection to the Sma/Mab signaling pathway, a TGF-β-like pathway in C. elegans, hints at potential involvement in development, body size regulation, or male tail formation . Alternatively, it may function as a modulator of signaling pathways through interactions with pathway components or through effects on protein phosphorylation states. Given its uncharacterized status, C34C12.7 could also represent a novel class of proteins with functions not yet described in the literature. Future research combining genetic, biochemical, and structural approaches will be essential to definitively establish the biological functions of this intriguing protein.

What technologies are emerging that could advance C34C12.7 research?

Emerging technologies offer promising avenues to accelerate research on uncharacterized proteins like C34C12.7. AlphaFold2 and other AI-based protein structure prediction tools can generate high-confidence structural models, providing insights into domain organization and potential functional sites without requiring experimental structure determination . CRISPR-Cas9 genome editing enables precise genetic manipulation, allowing researchers to create specific mutations, domain deletions, or tagged versions of C34C12.7 in its native genomic context in C. elegans. Single-cell RNA sequencing can reveal the expression pattern of C34C12.7 across different cell types and developmental stages, providing clues to its biological function.

Proximity labeling techniques such as TurboID or APEX2 can identify proteins that physically interact with or are in close proximity to C34C12.7 in living cells, helping to establish its protein interaction network . Advanced imaging approaches, including super-resolution microscopy and correlative light and electron microscopy, can precisely localize C34C12.7 within cellular structures. Cryo-electron tomography could visualize C34C12.7 in its native membrane environment, revealing structural details that might be lost in traditional purification approaches. Finally, multi-omics integration, combining transcriptomics, proteomics, and metabolomics data, can place C34C12.7 within broader cellular networks and pathways, generating new hypotheses about its function that can be tested experimentally.