Recombinant Nematostella vectensis UPF0443 protein v1g164247 (v1g164247)

What is the basic structural characterization of UPF0443 protein v1g164247?

The UPF0443 protein v1g164247 from Nematostella vectensis is a 62-amino acid protein classified as a single-pass membrane and coiled-coil domain-containing protein 4 homolog. The complete amino acid sequence is MRQLPGKAAKETRKMKRERKQQNKEGHNRVVTVAIPVCLAVFVMLIVYVYSATSKHRKWARR. Computational predictions suggest it contains a transmembrane region and potential coiled-coil motifs that could mediate protein-protein interactions .

How should recombinant v1g164247 protein be stored and reconstituted for optimal stability?

For optimal stability, recombinant v1g164247 protein should be stored at -20°C/-80°C upon receipt, with aliquoting recommended for multiple use to avoid repeated freeze-thaw cycles. The lyophilized protein should be briefly centrifuged before opening to bring contents to the bottom. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being the recommended default) before aliquoting is advised for long-term storage at -20°C/-80°C. For short-term use, working aliquots can be stored at 4°C for up to one week .

How can I validate the purity and identity of recombinant v1g164247 protein?

The purity of recombinant v1g164247 protein can be validated using SDS-PAGE, which should demonstrate >90% purity . For identity confirmation, western blotting using anti-His antibodies can verify the presence of the His-tag. Mass spectrometry-based approaches, including peptide mass fingerprinting or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), are recommended for definitive protein identification. These methods can be complemented with limited proteolysis to confirm the protein's structural integrity.

What are the optimal conditions for studying potential post-translational modifications of v1g164247?

For studying potential post-translational modifications (PTMs) of v1g164247, a comprehensive proteomic approach using immunoprecipitation followed by mass spectrometry would be optimal. Based on research methodologies applied to other proteins, researchers should consider both ubiquitin-like modifications and phosphorylation, as these are common regulatory mechanisms for membrane-associated proteins. The analysis should include enrichment steps specific to the PTM of interest, followed by high-resolution mass spectrometry .

For ubiquitin-like modifications:

• Perform immunoprecipitation under denaturing conditions

• Use antibodies specific to different ubiquitin-like modifiers (SUMO, NEDD8, etc.)

• Analyze by LC-MS/MS with specific search parameters for the PTM of interest

• Validate findings using site-directed mutagenesis of identified modification sites

How can I investigate the membrane topology and subcellular localization of v1g164247 in heterologous systems?

Investigating the membrane topology and subcellular localization of v1g164247 requires a multi-faceted approach:

• Computational prediction analysis:

  • Use topology prediction algorithms to identify transmembrane domains

  • Analyze signal peptides and targeting sequences

• Experimental verification:

  • Fluorescent protein tagging (N- and C-terminal) for live-cell imaging

  • Protease protection assays to determine membrane orientation

  • Immunocytochemistry with differential permeabilization techniques

  • Domain-specific antibody accessibility tests

• Subcellular fractionation:

  • Differential centrifugation followed by western blotting

  • Density gradient ultracentrifugation for membrane subfractionation

  • Co-localization with known organelle markers

This multi-method approach will provide complementary data to establish both the subcellular compartment(s) where v1g164247 resides and its orientation within the membrane.

What approaches can be used to identify potential interaction partners of v1g164247?

To identify potential interaction partners of v1g164247, researchers should implement multiple complementary techniques:

A thorough investigation would begin with co-immunoprecipitation followed by mass spectrometry to identify candidate interactors, which would then be validated using more targeted approaches such as pull-down assays and functional studies .

What strategies can be employed to improve the solubility of recombinant v1g164247 during expression and purification?

Improving the solubility of recombinant v1g164247, a membrane-associated protein, presents specific challenges that can be addressed through several strategies:

• Optimization of expression conditions:

  • Reduce expression temperature (16-20°C)

  • Use defined media with controlled induction

  • Co-express with molecular chaperones (GroEL/GroES, DnaK)

• Fusion tag selection:

  • Test solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Consider dual-tagging strategies for improved purification

• Buffer optimization during purification:

  • Include mild detergents (0.1% Triton X-100, CHAPS)

  • Add stabilizing agents (glycerol, arginine)

  • Use elevated salt concentrations (300-500 mM NaCl)

  • Test different pH conditions (pH 7.0-8.5)

• Extraction and solubilization:

  • For membrane-associated fraction, use detergent screening (DDM, LDAO, C12E8)

  • Consider bicelle or nanodisc reconstitution for downstream applications

These approaches should be systematically tested and optimized for v1g164247, with careful monitoring of protein quality at each step using analytical techniques such as dynamic light scattering and size exclusion chromatography.

How can I design functional assays to characterize the biological activity of v1g164247?

Designing functional assays for v1g164247 requires consideration of its predicted characteristics as a single-pass membrane protein. Without specific functional information available, a systematic approach should be implemented:

• Sequence-based function prediction:

  • Perform detailed bioinformatic analysis for functional domains

  • Identify conserved motifs across species

  • Predict potential phosphorylation sites or other regulatory elements

• Binding assays:

  • Develop solid-phase binding assays using purified protein

  • Screen against cellular extracts from N. vectensis tissues

  • Perform lipid binding assays to test membrane interactions

• Cell-based functional assays:

  • Generate stable cell lines expressing v1g164247

  • Assess effects on cellular phenotypes (proliferation, morphology)

  • Perform knockdown studies in native cells if available

• Structural characterization:

  • Circular dichroism spectroscopy for secondary structure assessment

  • Nuclear magnetic resonance (NMR) for structural determination of soluble domains

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

The results from these preliminary investigations will guide the development of more targeted functional assays based on the protein's predicted role.

What considerations are important when designing antibodies against v1g164247 for research applications?

Designing effective antibodies against v1g164247 requires careful epitope selection and validation strategies:

• Epitope analysis and selection:

  • Analyze the 62-amino acid sequence for antigenic regions using prediction algorithms

  • Avoid transmembrane domains, which are poorly immunogenic and less accessible

  • Consider sequence conservation if cross-reactivity with homologs is desired

  • Target N- or C-terminal regions that are likely more accessible

• Antibody development strategy:

  • Develop antibodies against multiple epitopes for redundancy

  • Consider both monoclonal (for specificity) and polyclonal (for sensitivity) approaches

  • Use synthetic peptides for immunization if full-length protein is difficult to express

• Validation requirements:

  • Test recognition of both native and denatured forms

  • Validate in multiple applications (Western blot, immunoprecipitation, immunofluorescence)

  • Include appropriate positive and negative controls

  • Perform knockdown/knockout validation for specificity

• Application-specific considerations:

  • For immunofluorescence, ensure accessibility of epitope in fixed cells

  • For immunoprecipitation, optimize buffer conditions for membrane protein solubilization

  • For flow cytometry, target extracellular epitopes if applicable

These considerations will increase the likelihood of developing useful antibody reagents for v1g164247 research.

How should differential expression analysis of v1g164247 across developmental stages be approached?

Differential expression analysis of v1g164247 across developmental stages should follow a structured approach similar to that used in transcriptomic studies of Nematostella vectensis:

• Experimental design considerations:

  • Include biological replicates (minimum 3-5 per developmental stage)

  • Control for batch effects and environmental variables

  • Include appropriate reference genes for normalization

• Quantification methods:

  • For transcript-level analysis, use RNA-Seq with appropriate depth (30M+ reads)

  • For protein-level analysis, use quantitative proteomics (TMT or SILAC labeling)

  • Process samples using standardized protocols to minimize technical variation

• Statistical analysis procedure:

  • Normalize expression data using appropriate methods (e.g., TPM for RNA-Seq)

  • Apply statistical tests suitable for time-course data, such as those implemented in DESeq2 or Sleuth

  • Use stringent significance thresholds (adjusted p-value < 0.05) and fold-change cutoffs

• Visualization and interpretation:

  • Generate heatmaps to visualize expression patterns across developmental stages

  • Perform clustering analysis to identify co-regulated genes

  • Compare expression patterns with known developmental markers

This approach will provide robust data on v1g164247 expression dynamics during development, enabling correlation with specific developmental processes.

What quality control metrics should be applied when analyzing mass spectrometry data for post-translational modifications of v1g164247?

When analyzing mass spectrometry data for post-translational modifications of v1g164247, several quality control metrics should be applied:

• Spectral quality assessment:

  • Signal-to-noise ratio (minimum 3:1 for reliable detection)

  • Mass accuracy (within 10 ppm for high-resolution instruments)

  • Fragment ion coverage (b and y ions covering >60% of the sequence)

  • Diagnostic ions specific to the modification (e.g., neutral loss patterns)

• Statistical validation:

  • False discovery rate control (typically <1% at peptide level)

  • Site localization probability scores (>0.75 for high confidence)

  • Require multiple unique peptides supporting each modification site

  • Use target-decoy approach to estimate false positive rate

• Biological validation:

  • Compare modification stoichiometry across biological replicates

  • Assess correlation with known biological stimuli

  • Validate key findings with orthogonal methods (e.g., site-directed mutagenesis)

• Data reporting standards:

  • Follow minimum information about a proteomics experiment (MIAPE) guidelines

  • Deposit raw data in public repositories

  • Report all search parameters and thresholds used

Adherence to these quality control metrics will ensure reliable identification of post-translational modifications on v1g164247.

How can phylogenetic analysis be used to understand the evolutionary context of v1g164247?

Phylogenetic analysis of v1g164247 can provide valuable insights into its evolutionary history and potential functional conservation. The approach should include:

• Sequence acquisition and alignment:

  • Perform BLAST searches against diverse taxonomic databases

  • Include sequences from representative species across metazoan phylogeny

  • Generate multiple sequence alignments using MAFFT or similar tools

  • Manually inspect and refine alignments, particularly for transmembrane regions

• Model selection and tree building:

  • Test alternative evolutionary models using ModelTest or similar tools

  • Implement maximum likelihood methods (RAxML, IQ-TREE)

  • Use Bayesian inference as a complementary approach

  • Apply appropriate gap handling strategies for sequences of different lengths

• Tree validation and evaluation:

  • Assess node support using bootstrap or approximate likelihood ratio tests

  • Compare trees generated using different methods and models

  • Consider gene structure and synteny data as complementary evidence

• Functional interpretation:

  • Map known functional domains onto the phylogenetic tree

  • Analyze patterns of sequence conservation and diversification

  • Identify lineage-specific duplications or losses

  • Correlate evolutionary patterns with species-specific adaptations

This comprehensive phylogenetic approach will place v1g164247 in its proper evolutionary context and may suggest functional hypotheses based on patterns of conservation.

What controls should be included when performing localization studies with tagged v1g164247 constructs?

When performing localization studies with tagged v1g164247 constructs, the following controls should be included:

• Tag-position controls:

  • Compare N-terminal and C-terminal tagged versions

  • Include a middle-insertion tag if feasible

  • Test untagged protein with specific antibodies if available

• Expression level controls:

  • Include both stable low-expression and transient systems

  • Use inducible promoters to test expression-dependent effects

  • Compare to endogenous levels if possible using quantitative methods

• Specificity controls:

  • Empty vector expressing tag alone

  • Irrelevant protein with same tag

  • Mutant v1g164247 with altered targeting sequences

• Technical controls:

  • Co-localization markers for specific subcellular compartments

  • Live/fixed cell comparisons to rule out fixation artifacts

  • Multiple cell types to confirm consistency of localization pattern

• Functional validation:

  • Rescue experiments with wild-type protein if knockdown phenotype is available

  • Structured illumination or super-resolution microscopy for detailed localization

These comprehensive controls will ensure that any observed localization patterns are reliable and biologically relevant.

How can I design experiments to investigate the potential role of v1g164247 in membrane signaling pathways?

To investigate the potential role of v1g164247 in membrane signaling pathways, a systematic experimental approach should be implemented:

• Interactome mapping:

  • Perform proximity labeling experiments (BioID, APEX) in relevant cell types

  • Conduct co-immunoprecipitation studies with and without crosslinking

  • Screen for interactions with known signaling components

• Signal transduction analysis:

  • Overexpress or knock down v1g164247 and monitor effects on:

    • Second messenger levels (cAMP, Ca²⁺, IP₃)

    • Phosphorylation status of downstream effectors

    • Transcriptional responses of pathway-specific reporters

• Structure-function analysis:

  • Generate deletion constructs to identify functional domains

  • Perform site-directed mutagenesis of predicted interaction motifs

  • Create chimeric proteins to test domain-specific functions

• Physiological stimulation:

  • Expose cells expressing v1g164247 to physiologically relevant stimuli

  • Monitor protein dynamics using real-time imaging techniques

  • Assess post-translational modification changes in response to stimulation

• Systems-level analysis:

  • Integrate data from these approaches using network analysis

  • Develop testable hypotheses about pathway involvement

  • Design targeted validation experiments for key findings

This comprehensive approach will provide multiple lines of evidence regarding the role of v1g164247 in membrane signaling pathways.