Recombinant Uncharacterized protein C18B2.1 (C18B2.1)

What are the optimal storage and reconstitution conditions for C18B2.1?

For optimal research results, C18B2.1 should be stored as follows:

For reconstitution:

• Centrifuge the vial briefly before opening to collect contents at the bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage

• Aliquot and store at -20°C/-80°C

Repeated freeze-thaw cycles significantly reduce protein stability and should be avoided to maintain experimental consistency.

What expression systems are commonly used for C18B2.1 production?

The standard expression system for C18B2.1 is E. coli, which provides several methodological advantages for research applications:

• High yield of recombinant protein

• Cost-effective production

• Simplified purification via the N-terminal His tag

• Compatibility with standard protein purification techniques

What bioinformatic approaches can predict structural and functional characteristics of C18B2.1?

For initial characterization of uncharacterized proteins like C18B2.1, a systematic computational pipeline is recommended:

• Physicochemical characterization:

  • Calculate instability index (II) to assess protein stability

  • Determine theoretical isoelectric point (pI)

  • Evaluate GRAVY (Grand Average of Hydropathy) values to determine polarity

  • For proteins similar to those in search result , approximately 70% have negative GRAVY values, indicating non-polar characteristics

• Subcellular localization prediction:

  • Use PSORTb to predict cellular compartmentalization

  • For hypothetical proteins (HPs) similar to C18B2.1, distribution typically follows:

    • 32-38% cytoplasmic

    • 23-27% cytoplasmic membrane

    • 1-3% extracellular

    • 37-40% unknown location

• Secretory nature prediction:

  • Apply SignalP 5.0 for secretion signal detection

  • In comparable HPs, 87-92% are typically non-secretory

How can conserved domains in C18B2.1 be identified and analyzed?

The identification of conserved domains provides critical insights into potential function:

• Domain identification methodology:

  • Use NCBI Batch CDD (Conserved Domain Database) search

  • Apply HMMer for profile hidden Markov model analysis

  • Perform InterProScan for integrated protein signature recognition

• Analysis of identified domains:

  • Compare with known functional signatures from superfamilies such as:

    • Beta_helix

    • HDC_protein

    • M34_PPEP

    • SPASM

    • Other domains found in similar uncharacterized proteins

• Functional prediction:

  • Correlate domains with known biochemical functions

  • Predict possible enzymatic activities based on conserved catalytic residues

  • Assess structural similarities to characterized proteins

What experimental approaches are most effective for functional characterization of C18B2.1?

A multi-tiered experimental design is recommended for comprehensive functional characterization:

Each approach should be implemented sequentially, with results from initial studies informing the design of subsequent experiments to maximize research efficiency.

How can homology modeling enhance understanding of C18B2.1 structure and function?

For uncharacterized proteins like C18B2.1, homology modeling provides a methodological framework to predict structure-function relationships:

• Template selection criteria:

  • Identify proteins with similar sequence or domain architecture

  • Prioritize templates with experimental structural data

  • Evaluate sequence identity (optimal >30%) and coverage

• Modeling workflow:

  • Generate multiple alignments with potential templates

  • Build initial models using platforms like SWISS-MODEL, Phyre2, or I-TASSER

  • Refine models through energy minimization

  • Validate using Ramachandran plots, QMEAN, and other quality metrics

• Functional inference:

  • Identify potential binding pockets or catalytic sites

  • Map conserved residues onto the structural model

  • Predict protein-protein or protein-ligand interactions

  • Design site-directed mutagenesis experiments to validate predictions

What are the challenges in determining if C18B2.1 has therapeutic or vaccine potential?

Evaluating uncharacterized proteins for therapeutic applications requires systematic assessment of several parameters:

• Human homology assessment:

  • Conduct BLAST analysis against human proteome

  • Apply stringent criteria: ≥35% identity, query coverage ≥35%, E-value <10e-5

  • For similar HPs, approximately 99.7% are typically non-homologous to human proteins

• Virulence factor analysis:

  • Use specialized prediction tools (VirulentPred, VICMpred)

  • Examine subcellular localization for potential drug accessibility

  • For similar HPs, only 0.25-1.45% typically show virulence characteristics

• Antigenicity and allergenicity evaluation:

  • Apply VaxiJen server for antigenicity prediction

  • Use AllerTOP for allergenicity assessment

  • Similar HPs show 36-41% antigenicity rates with minimal allergenicity

• Experimental validation challenges:

  • Limited knowledge of natural function complicates therapeutic targeting

  • Potential off-target effects require extensive testing

  • Animal model selection for an uncharacterized protein presents significant challenges

How can protein expression and purification of C18B2.1 be optimized for structural studies?

Optimizing recombinant C18B2.1 production for structural studies requires attention to several methodological details:

• Expression optimization:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Evaluate induction conditions (IPTG concentration, temperature, duration)

  • Consider codon optimization for C. elegans sequences

  • Explore fusion partners beyond His-tag (MBP, GST) to enhance solubility

• Purification refinement:

  • Implement multi-step purification strategy:

    • Initial IMAC (Immobilized Metal Affinity Chromatography) using His-tag

    • Secondary ion exchange chromatography

    • Final size exclusion chromatography for highest purity

  • Optimize buffer composition for protein stability

• Quality assessment:

  • Verify purity by SDS-PAGE (>95% for structural studies)

  • Confirm identity by mass spectrometry

  • Assess homogeneity by dynamic light scattering

  • Validate proper folding using circular dichroism

What experimental approaches can resolve contradictory functional predictions for C18B2.1?

When computational predictions yield conflicting functional hypotheses, a systematic experimental approach is required:

• Prioritization of hypotheses:

  • Rank predictions based on confidence scores

  • Consider evolutionary conservation of predicted functions

  • Evaluate structural compatibility with predicted activities

• Targeted validation experiments:

  • Design specific biochemical assays for each predicted function

  • Develop activity panels to test multiple potential functions

  • Create focused mutant libraries targeting predicted functional residues

• Unbiased functional screening:

  • Perform metabolite profiling in knockout/overexpression systems

  • Conduct phenotype microarrays to identify growth conditions affected

  • Implement global interaction screens (genetic and physical)

• Results integration framework:

  • Create weighted scoring system for experimental evidence

  • Apply Bayesian analysis to update functional probability assessments

  • Develop a decision tree for subsequent experimental design

How can researchers effectively utilize C18B2.1 in comparative studies with other uncharacterized proteins?

Comparative analysis of uncharacterized proteins requires rigorous methodology to generate meaningful insights:

• Selection of comparison cohort:

  • Identify proteins with similar:

    • Domain architecture

    • Phylogenetic distribution

    • Expression patterns

    • Predicted subcellular localization

• Standardized characterization protocol:

  • Apply identical experimental conditions across all proteins

  • Utilize consistent bioinformatic tools and parameters

  • Implement parallel functional assays

• Data integration and visualization:

  • Create similarity networks based on multiple parameters

  • Develop hierarchical clustering of functional predictions

  • Generate phylogenetic profiles with mapped functional characteristics

• Inference methodology:

  • Apply guilt-by-association principles for functional annotation

  • Utilize machine learning to identify patterns across datasets

  • Implement Bayesian networks to predict functional relationships

How can systems biology approaches enhance understanding of C18B2.1 function in C. elegans?

Integrating C18B2.1 research into systems-level analysis provides contextual understanding of its biological role:

• Multi-omics integration methodology:

  • Correlate transcriptomics data showing C18B2.1 expression patterns

  • Analyze proteomics datasets for co-expressed proteins

  • Examine metabolomics changes in C18B2.1 mutants

  • Create integrated networks incorporating all data types

• Network analysis techniques:

  • Implement weighted gene co-expression network analysis (WGCNA)

  • Construct protein-protein interaction networks with C18B2.1

  • Apply Bayesian network inference to identify causal relationships

  • Utilize random forest algorithms to predict functional associations

• Phenotypic profiling:

  • Conduct systematic RNAi screens in different genetic backgrounds

  • Perform high-content imaging of C18B2.1 mutants

  • Implement automated behavioral analysis of C. elegans strains

  • Create comprehensive phenotypic signatures for comparative analysis

What emerging technologies are most promising for characterizing proteins like C18B2.1?

Recent technological advances offer new opportunities for uncharacterized protein research:

• AlphaFold2 and other AI-based structural prediction:

  • Generate high-confidence structural models without experimental data

  • Predict protein-protein interactions based on structural complementarity

  • Identify potential binding sites and functional regions

• Single-cell technologies:

  • Apply single-cell transcriptomics to identify cell types expressing C18B2.1

  • Implement spatial transcriptomics to map expression patterns

  • Utilize single-cell proteomics to measure protein levels in specific cells

• CRISPR-based functional genomics:

  • Perform CRISPR activation/interference screens

  • Implement base editing for specific amino acid substitutions

  • Utilize prime editing for precise genetic modifications

  • Develop CRISPR-based synthetic genetic interaction mapping