Recombinant UPF0392 protein F59C6.8 (F59C6.8)

What is UPF0392 protein F59C6.8 and why is it significant for research?

UPF0392 protein F59C6.8 is an uncharacterized protein from Caenorhabditis elegans, classified in the UPF0392 protein family. Its significance stems from its potential role in developmental biology and comparative proteomics. As an uncharacterized protein family (UPF) member, it represents an opportunity for novel discovery in C. elegans biology. The protein consists of 515 amino acids and is available as a recombinant protein for research applications . The "UPF" designation indicates that while the protein's sequence is known, its biological function remains largely undetermined, making it a valuable target for fundamental research in molecular biology and nematode physiology.

How should recombinant UPF0392 protein F59C6.8 be stored and handled to maintain stability?

For optimal stability and activity, recombinant UPF0392 protein F59C6.8 should be stored according to the following guidelines:

• Long-term storage: Store at -20°C or -80°C for extended periods

• Buffer composition: The protein is typically supplied in Tris-based buffer with 50% glycerol, optimized for this specific protein

• Working aliquots: Store at 4°C for up to one week to minimize freeze-thaw cycles

• Freeze-thaw considerations: Repeated freezing and thawing is not recommended as it can lead to protein degradation and loss of activity

When preparing working solutions, researchers should reconstitute lyophilized protein according to manufacturer specifications. Unlike some recombinant proteins that benefit from carrier proteins like BSA, specific carrier requirements for F59C6.8 may vary based on the experimental application and should be determined empirically.

What are the recommended approaches for studying the function of an uncharacterized protein like F59C6.8?

To elucidate the function of UPF0392 protein F59C6.8, a multi-faceted approach is recommended:

• Comparative sequence analysis: Utilize bioinformatics tools (BLAST, HMMer, InterPro) to identify homologs in other organisms and predict functional domains based on evolutionary conservation

• Protein-protein interaction studies: Employ yeast two-hybrid, co-immunoprecipitation, or proximity labeling methods to identify binding partners. The empty "Interacting Protein" section in available databases suggests this work is still needed

• Expression pattern analysis: Characterize temporal and spatial expression patterns using techniques such as RNA-seq, qPCR, and in situ hybridization in C. elegans at different developmental stages

• Loss-of-function studies: Generate knockout or knockdown C. elegans strains (using CRISPR-Cas9 or RNAi) to observe phenotypic consequences

• Structural biology approaches: Perform X-ray crystallography, NMR, or cryo-EM studies to determine three-dimensional structure, which can provide function insights

• Subcellular localization: Use fluorescently tagged versions of the protein to determine its location within cells, providing clues about potential functions

Each of these approaches provides complementary information that, when integrated, can reveal the biological role of this uncharacterized protein.

How can I design experiments to determine if F59C6.8 is involved in specific cellular pathways?

To investigate pathway involvement of UPF0392 protein F59C6.8, consider the following experimental design strategy:

• Pathway-specific perturbation: Expose C. elegans to conditions that activate or inhibit specific pathways of interest, then measure changes in F59C6.8 expression or modification state

• Genetic interaction screens: Cross F59C6.8 mutant strains with strains carrying mutations in known pathway components to identify synthetic phenotypes that suggest pathway relationships

• Phosphoproteomics analysis: Determine if F59C6.8 undergoes phosphorylation in response to specific signals, as many pathway proteins are regulated by phosphorylation cascades

• Transcriptional profiling: Compare gene expression profiles between wild-type and F59C6.8 mutant worms to identify affected pathways

• Metabolomics approaches: Analyze metabolite changes in F59C6.8 mutants to identify affected biochemical pathways

The experimental design should include:

This systematic approach can reveal pathways influenced by F59C6.8, despite the current lack of annotated pathway information in databases .

What expression systems are optimal for producing functional recombinant UPF0392 protein F59C6.8?

Based on current research protocols, the following expression systems have been evaluated for producing recombinant UPF0392 protein F59C6.8:

• E. coli expression system: Currently the most documented system for F59C6.8 production, using His-tagged constructs. This system can produce the full-length protein (1-515 amino acids) . Advantages include high yield and cost-effectiveness, though proper folding may be a concern for complex eukaryotic proteins.

• Insect cell expression: While not explicitly documented for F59C6.8 in the provided information, this system can provide eukaryotic post-translational modifications that may be important for function.

• Mammalian expression systems: For studies requiring mammalian post-translational modifications or when protein misfolding occurs in prokaryotic systems.

Optimization considerations for expression include:

• Codon optimization for the selected expression host

• Selection of appropriate fusion tags (His, GST, MBP) to enhance solubility and facilitate purification

• Inclusion of protease cleavage sites to remove tags post-purification

• Expression temperature and induction conditions to balance yield with proper folding

• Buffer composition during purification to maintain stability and activity

For functional studies, researchers should verify protein folding and activity following purification, as improper folding can occur in heterologous expression systems, especially for proteins with predicted transmembrane regions like F59C6.8.

How can structural prediction tools be applied to gain insights into F59C6.8 function when crystallographic data is unavailable?

In the absence of crystallographic data for UPF0392 protein F59C6.8, modern computational approaches can provide valuable structural insights:

• AlphaFold2 and RoseTTAFold analysis: These AI-based structure prediction tools have revolutionized protein structure prediction and can generate highly accurate models, especially for domains with homology to known structures

• Molecular dynamics simulations: Apply MD simulations to predicted structures to identify stable conformations and potential ligand-binding pockets

• Threading and homology modeling: Identify structural templates from proteins with similar sequences and generate models based on these templates

• Evolutionary coupling analysis: Methods like EVfold use evolutionary constraints inferred from multiple sequence alignments to predict residue-residue contacts

• Integrative modeling approaches: Combine low-resolution experimental data (such as crosslinking mass spectrometry or small-angle X-ray scattering) with computational predictions

When interpreting computational models, researchers should:

• Assess model confidence metrics provided by prediction software

• Validate predictions against any available experimental data

• Focus analysis on regions with higher prediction confidence

• Compare predictions from multiple independent methods

These predictions can guide experimental design by identifying potential functional sites, binding interfaces, and structural features that might not be apparent from sequence analysis alone.

What are the current hypotheses regarding F59C6.8's role in C. elegans development or physiology?

While specific functional information for UPF0392 protein F59C6.8 remains limited, several hypotheses can be formulated based on sequence analysis, structural features, and knowledge of related proteins:

• Membrane-associated signaling: The hydrophobic regions in the N-terminal sequence suggest possible membrane association, potentially involved in signal transduction pathways during development

• Developmental regulation: Many UPF family proteins later characterized have been found to play roles in developmental processes, particularly in tissue morphogenesis

• Stress response modulator: Several uncharacterized proteins in C. elegans have been subsequently identified as stress response factors, particularly in oxidative stress or heat shock pathways

• Metabolic function: The protein may function in metabolic pathways specific to nematode development or homeostasis

To test these hypotheses, researchers could employ:

• Temporal expression analysis during C. elegans development

• Tissue-specific knockout studies

• Stress challenge experiments comparing wild-type and F59C6.8 mutant worms

• Metabolic flux analysis under various conditions

The current lack of pathway annotation suggests that traditional bioinformatic approaches have been insufficient to classify this protein's function, highlighting the need for direct experimental investigation.

How can mass spectrometry techniques be optimized to study post-translational modifications of F59C6.8?

Optimizing mass spectrometry (MS) for studying post-translational modifications (PTMs) of UPF0392 protein F59C6.8 requires specialized approaches:

• Sample preparation optimization:

  • Enrich for specific PTMs using antibodies or chemical approaches (e.g., TiO₂ for phosphopeptides)

  • Apply multiple proteases beyond trypsin (e.g., chymotrypsin, AspN) to improve sequence coverage

  • Use native purification conditions to preserve labile modifications

• MS acquisition strategies:

  • Employ electron transfer dissociation (ETD) or electron capture dissociation (ECD) to preserve labile modifications

  • Use parallel reaction monitoring (PRM) for targeted analysis of predicted modification sites

  • Apply data-independent acquisition (DIA) to improve detection of low-abundance modified peptides

• Data analysis considerations:

  • Search for multiple potential modifications simultaneously (phosphorylation, acetylation, methylation, ubiquitination)

  • Validate PTM site localization using site-determining ions and statistical approaches

  • Consider quantitative approaches to measure stoichiometry of modifications

Given the potential membrane association of F59C6.8, special attention should be paid to extraction methods that effectively solubilize the protein while preserving modifications.

What are common challenges in expressing and purifying recombinant UPF0392 protein F59C6.8, and how can they be addressed?

Researchers working with recombinant UPF0392 protein F59C6.8 may encounter several challenges during expression and purification:

• Protein solubility issues:

  • Challenge: The hydrophobic regions in F59C6.8 may cause aggregation during expression

  • Solution: Optimize expression temperature (try 18°C instead of 37°C), use solubility-enhancing tags (MBP, SUMO), or add mild detergents during lysis

• Low expression yield:

  • Challenge: Heterologous expression of C. elegans proteins can result in low yields

  • Solution: Optimize codon usage for the expression host, test different promoter systems, or increase culture volume

• Protein misfolding:

  • Challenge: Improper folding affecting structure and function

  • Solution: Co-express with chaperones, use slower induction protocols, or consider refolding from inclusion bodies

• Proteolytic degradation:

  • Challenge: Sensitivity to proteases during purification

  • Solution: Add protease inhibitors, reduce purification time, or identify and mutate protease-sensitive sites

• Aggregation during storage:

  • Challenge: Protein aggregation after purification

  • Solution: Optimize buffer composition (add glycerol as indicated in standard protocols ), determine optimal pH, or add stabilizing agents

When troubleshooting purification issues, researchers should conduct small-scale expression trials with multiple conditions before scaling up. Additionally, quality control testing using size exclusion chromatography or dynamic light scattering can help identify aggregation problems early in the process.

How can researchers validate antibody specificity for studies involving F59C6.8?

Ensuring antibody specificity is critical for reliable results when studying UPF0392 protein F59C6.8. A comprehensive validation protocol includes:

• Positive and negative controls:

  • Use recombinant F59C6.8 protein as a positive control

  • Include lysates from F59C6.8 knockout C. elegans strains as negative controls

  • Test antibody on closely related proteins to assess cross-reactivity

• Multiple antibody validation approaches:

  • Western blotting with appropriate size controls and blocking peptides

  • Immunoprecipitation followed by mass spectrometry identification

  • Immunofluorescence with parallel siRNA knockdown validation

• Peptide competition assays:

  • Pre-incubate antibody with excess synthetic peptide from the immunogen

  • Compare signal intensity between competed and non-competed samples

• Orthogonal validation methods:

  • Compare protein expression patterns using multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Use tagged versions of the protein as parallel validation

• Quantitative assessment metrics:

  • Calculate signal-to-noise ratios across different antibody concentrations

  • Document the linear dynamic range of detection

  • Assess batch-to-batch variability when using multiple antibody lots

This rigorous validation approach ensures that experimental observations truly reflect F59C6.8 biology rather than antibody artifacts or cross-reactivity with related proteins.

What strategies can overcome challenges in identifying interaction partners of F59C6.8?

Identifying interaction partners of poorly characterized proteins like UPF0392 protein F59C6.8 presents unique challenges. The following strategies can help overcome these obstacles:

• Proximity-dependent labeling approaches:

  • BioID or TurboID fusion constructs expressed in C. elegans to label proximal proteins in vivo

  • APEX2-based proximity labeling for temporal control of labeling reactions

  • These methods are particularly valuable for transient or weak interactions that may be lost in traditional co-IP approaches

• Cross-linking mass spectrometry (XL-MS):

  • Apply protein cross-linkers of various lengths to stabilize interactions

  • MS/MS analysis to identify cross-linked peptides, revealing interaction interfaces

  • In vivo cross-linking in C. elegans for physiologically relevant conditions

• Split-reporter protein complementation assays:

  • Split-GFP, split-luciferase, or split-ubiquitin systems to test candidate interactions

  • Library screening approaches to identify novel interaction partners

• Affinity purification optimization:

  • Test multiple tagging strategies (N-terminal vs. C-terminal tags)

  • Optimize lysis and binding conditions (detergents, salt concentration, pH)

  • Consider native purification conditions to preserve physiological complexes

• Functional genomics correlation approaches:

  • Genetic interaction mapping to identify functional relationships

  • Co-expression analysis across developmental stages or conditions

  • Comparative interactomics using data from related proteins in other species

Each approach has strengths and limitations, so researchers should consider combining multiple methods for comprehensive interaction mapping, particularly given the current lack of annotated interaction partners for F59C6.8 .

How can genome editing techniques be applied to study F59C6.8 function in vivo?

CRISPR-Cas9 and other genome editing technologies offer powerful approaches to studying UPF0392 protein F59C6.8 function directly in C. elegans:

• Knockout generation strategies:

  • Complete gene deletion to assess loss-of-function phenotypes

  • Introduction of early stop codons or frameshift mutations

  • Conditional knockouts using tissue-specific or inducible Cre-lox systems

• Endogenous tagging approaches:

  • C-terminal or N-terminal fluorescent protein fusions for localization studies

  • Addition of affinity tags (FLAG, HA, BioID) for interaction studies

  • Introduction of degron tags for rapid protein depletion

• Domain-specific mutations:

  • Targeted mutation of predicted functional residues to assess their importance

  • Generation of phospho-mimetic or phospho-dead mutations at predicted modification sites

  • Deletion of specific protein domains to determine their contribution to function

• Humanized worm models:

  • Replace F59C6.8 with human homologs (if identified) to assess functional conservation

  • Create chimeric proteins to map functional domains across species

• Reporter gene insertion:

  • Insert fluorescent reporters under the control of the endogenous F59C6.8 promoter

  • Create transcriptional and translational fusions to study expression patterns

When designing genome editing experiments, researchers should carefully consider:

• Potential off-target effects and include appropriate controls

• The impact of modifications on protein folding and stability

• Phenotypic assays relevant to predicted functions

• The need for homozygous vs. heterozygous mutations

These approaches can provide definitive evidence of F59C6.8 function that complements biochemical and structural studies.

What are promising research directions for elucidating the potential role of F59C6.8 in disease models?

While UPF0392 protein F59C6.8 is a C. elegans protein, investigating its potential relevance to disease models offers several promising research directions:

• Comparative genomics approaches:

  • Identify human homologs or proteins with similar domain architecture

  • Analyze conservation of key residues across species

  • Examine whether human homologs are associated with disease phenotypes

• C. elegans disease modeling:

  • Investigate F59C6.8 function in worm models of neurodegeneration, aging, or metabolic disorders

  • Assess whether F59C6.8 modulates phenotypes in established C. elegans disease models

  • Screen for genetic interactions with known disease-associated genes

• Stress response studies:

  • Determine if F59C6.8 expression or localization changes under stress conditions

  • Test whether F59C6.8 mutants show altered sensitivity to various stressors

  • Investigate potential roles in proteostasis or oxidative stress response

• Developmental phenotyping:

  • Characterize developmental defects in F59C6.8 mutants that might parallel human developmental disorders

  • Assess cell fate decisions and morphogenesis in the absence of F59C6.8

• Drug discovery applications:

  • Utilize F59C6.8 mutant phenotypes for small molecule screening

  • Identify compounds that suppress mutant phenotypes as potential therapeutic leads

  • Develop assays to screen for modulators of F59C6.8 function or expression

These approaches take advantage of C. elegans as a powerful model organism while establishing connections to potential human disease relevance, even for a currently uncharacterized protein.

How can systems biology approaches integrate F59C6.8 into broader protein networks and functional modules?

Systems biology offers powerful frameworks for positioning UPF0392 protein F59C6.8 within the broader context of C. elegans biology:

• Network integration approaches:

  • Construct protein-protein interaction networks incorporating F59C6.8 and its partners

  • Apply graph theory algorithms to predict functional modules containing F59C6.8

  • Use Bayesian network analysis to infer causal relationships in regulatory networks

• Multi-omics data integration:

  • Correlate F59C6.8 expression with transcriptome, proteome, and metabolome data

  • Identify conditions or developmental stages where F59C6.8 co-expresses with functionally characterized genes

  • Apply dimensionality reduction techniques to position F59C6.8 within the broader functional landscape

• Phenotypic profiling:

  • Compare F59C6.8 mutant phenotypes with phenotypic signatures of other gene perturbations

  • Employ machine learning to classify F59C6.8 into functional categories based on phenotypic similarity

  • Develop quantitative phenotyping approaches for subtle phenotypes

• Evolutionary systems biology:

  • Analyze the evolutionary history of the UPF0392 family across species

  • Identify evolutionary rate changes that might indicate functional constraints

  • Map synteny relationships to identify conserved genomic neighborhoods

• Predictive modeling:

  • Develop mathematical models incorporating F59C6.8 into relevant biological processes

  • Simulate perturbation effects to generate testable hypotheses

  • Apply flux balance analysis if metabolic functions are suspected

These integrative approaches can contextualize F59C6.8 within broader biological systems, potentially revealing functions that may not be apparent from focused molecular studies alone.