Recombinant Pongo abelii Uncharacterized protein C4orf3 homolog

What is Pongo abelii Uncharacterized protein C4orf3 homolog and what are its key molecular characteristics?

The Pongo abelii Uncharacterized protein C4orf3 homolog is a protein found in Sumatran orangutans (Pongo abelii) that is homologous to the human C4orf3 (chromosome 4 open reading frame 3) protein. This is a relatively small protein consisting of 66 amino acids with the full sequence: MEVDVPGVDGRDGLRERRGLSEGGRQNLDVRPQSGANGLPKHSYWLDLWLFIFFDVVVFLFVYFLP . In database nomenclature, it is identified by UniProt accession number Q5R4B0 . The gene encoding this protein is referred to as C4H4orf3 (chromosome 4 C4orf3 homolog) in the orangutan genome, as indicated in mRNA accession NM_001133392.1 .

To determine its key molecular characteristics, researchers would typically employ multiple approaches including:

• Amino acid composition analysis to identify hydrophobic/hydrophilic regions

• Secondary structure prediction software to determine α-helices, β-sheets, and random coils

• Bioinformatic analysis for post-translational modification sites and functional domains

What is currently known about the structural characteristics of this protein?

• Sequence analysis reveals a 66-amino acid protein with potential membrane-spanning regions based on the presence of hydrophobic residues (WLFIFFDVVVFLFVYFLP at the C-terminus) .

• Secondary structure prediction tools would likely identify potential α-helices and β-sheets.

• Homology modeling against better-characterized homologs may provide initial structural insights.

The relatively high proportion of hydrophobic residues in the C-terminal portion suggests possible membrane association or interaction, though experimental validation is required. For detailed structural characterization, researchers would need to pursue X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy studies.

What are the optimal storage and handling conditions for this recombinant protein?

Based on established protocols for this protein, the following storage and handling conditions are recommended:

• Storage buffer: Tris-based buffer containing 50% glycerol, specifically optimized for this protein .

• Primary storage temperature: -20°C for regular use, or -80°C for extended long-term storage .

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

• Handling precautions: Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of biological activity .

For experiments requiring high protein stability, preparation of single-use aliquots upon initial thawing is recommended. Additionally, researchers should verify protein integrity after extended storage through SDS-PAGE or other analytical methods.

What expression systems have been successfully used for producing this recombinant protein?

The recombinant Pongo abelii Uncharacterized protein C4orf3 homolog has been successfully expressed in E. coli expression systems, as documented in commercial preparations . For researchers designing expression strategies:

• Bacterial expression (E. coli): The most widely documented system, suitable for producing this relatively small protein (66 amino acids) with His-tagging for purification .

• Alternative expression systems to consider:

  • Yeast expression systems (S. cerevisiae or P. pastoris) for potential post-translational modifications

  • Mammalian cell expression for studying protein in a more native-like environment

  • Cell-free protein synthesis for rapid production without cellular constraints

The selection of an appropriate expression system should be guided by:

• Experimental objectives (structural studies vs. functional assays)

• Required protein yield

• Need for post-translational modifications

• Downstream application requirements

What purification strategies are recommended for obtaining high-purity preparations of this protein?

For purification of Recombinant Pongo abelii Uncharacterized protein C4orf3 homolog, a multi-step purification strategy is recommended:

• Affinity chromatography: Utilizing His-tag affinity, as commercially available versions of this protein are His-tagged . This provides initial purification with relatively high specificity.

• Size exclusion chromatography: Given the small size of the protein (66 amino acids), this serves as an excellent secondary purification step to separate the target protein from larger contaminants.

• Ion exchange chromatography: Based on the protein's theoretical isoelectric point, this can provide additional purification.

A typical purification workflow would include:

• Cell lysis using appropriate buffer systems (typically containing protease inhibitors)

• Clarification of lysate by centrifugation (15,000×g for 20 minutes at 4°C)

• IMAC purification using Ni-NTA or similar resin

• Buffer exchange and concentration

• Final polishing step using size exclusion or ion exchange chromatography

Protein purity should be assessed at each stage using SDS-PAGE and potentially Western blotting with anti-His antibodies.

What analytical methods should be employed to verify identity and structural integrity of the purified protein?

To verify the identity and structural integrity of purified Recombinant Pongo abelii Uncharacterized protein C4orf3 homolog, researchers should implement a multi-technique analytical approach:

• SDS-PAGE: For molecular weight confirmation and initial purity assessment.

• Western Blotting: Using anti-His antibodies (for tagged versions) or specific antibodies against the protein.

• Mass Spectrometry:

  • MALDI-TOF for accurate molecular weight determination

  • LC-MS/MS for peptide mapping and sequence verification, similar to proteomics approaches described in related studies

• Circular Dichroism (CD): To assess secondary structure elements and protein folding.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): For determining oligomeric state and homogeneity.

For comprehensive characterization, a combination of the above techniques should be employed. The proteomics workflow might follow similar parameters to those described in related research: using tools like Proteome Discoverer 1.3 with search parameters set for trypsin digestion and database matching against appropriate taxonomic databases .

What experimental approaches can be employed to elucidate the function of this uncharacterized protein?

Given the uncharacterized nature of this protein, a multi-faceted experimental approach is required to establish its function:

• Bioinformatic analysis:

  • Sequence homology comparisons with characterized proteins

  • Domain prediction and motif scanning

  • Gene ontology term prediction based on structural features

• Interactome analysis:

  • Yeast two-hybrid screening to identify interaction partners

  • Co-immunoprecipitation followed by mass spectrometry

  • Protein microarray studies

• Cellular localization studies:

  • Fluorescent tagging and microscopy

  • Subcellular fractionation followed by Western blotting

• Loss-of-function studies:

  • CRISPR-Cas9 knockout in appropriate cell lines

  • RNA interference (similar to approaches used in related studies)

  • Analysis of resulting phenotypes and pathway disruptions

• Gain-of-function studies:

  • Overexpression analysis

  • Rescue experiments in knockout systems

As demonstrated in related protein studies, quantitative real-time PCR can be utilized to analyze gene expression changes in response to perturbation of the protein of interest .

How can researchers investigate potential involvement of this protein in post-translational modification pathways?

To investigate potential involvement of the Pongo abelii C4orf3 homolog in post-translational modification pathways, particularly ubiquitination which is relevant based on related studies , researchers should consider:

• Ubiquitination analysis:

  • Immunoprecipitation followed by ubiquitin-specific Western blotting

  • Mass spectrometry analysis to identify ubiquitination sites

  • In vitro ubiquitination assays with E1, E2, and candidate E3 ligases

• Proteasome interaction studies:

  • Proteasome inhibition experiments

  • Co-immunoprecipitation with proteasome components

  • Protein stability assays in the presence of proteasome inhibitors

• E3 ligase interaction screening:

  • Focused screening with RING finger ubiquitin ligases, including the SIAH family

  • Validation of interactions through biochemical and cellular assays

• Proteomics approach:

  • iTRAQ or TMT-based quantitative proteomics to identify changes in the ubiquitinome upon manipulation of C4orf3 homolog expression

  • Similar to the approach described in the SIAH1 study, where RNAi knockdown followed by LC-MS/MS analysis revealed changes in ubiquitinated proteins

These approaches would help determine whether the protein functions as a substrate, regulator, or component of ubiquitination pathways, which appears to be a relevant direction based on studies of related proteins.

What strategies can be used to investigate evolutionary conservation and divergence of function for this protein?

To investigate evolutionary conservation and divergence of the C4orf3 homolog protein, researchers should employ a comparative genomics and functional analysis approach:

• Sequence analysis and phylogenetics:

  • Multiple sequence alignment of C4orf3 homologs across primate species

  • Calculation of sequence conservation scores for individual residues

  • Phylogenetic tree construction to map evolutionary relationships

  • Identification of positively selected sites using dN/dS ratio analysis

• Structural comparison:

  • Homology modeling of C4orf3 homologs from different species

  • Superimposition of predicted structures to identify conserved structural features

• Functional conservation testing:

  • Cross-species complementation experiments

  • Expression of homologs from different species in knockout cell lines

  • Assessment of functional rescue to determine functional equivalence

• Expression pattern analysis:

  • Comparative transcriptomics to evaluate tissue-specific expression across species

  • Analysis of regulatory regions to identify conserved and divergent control elements

This approach would help establish whether the uncharacterized function of this protein is conserved across primate evolution or has undergone divergence in the orangutan lineage.

How might this protein relate to membrane dynamics based on its sequence characteristics?

The amino acid sequence of Pongo abelii Uncharacterized protein C4orf3 homolog (MEVDVPGVDGRDGLRERRGLSEGGRQNLDVRPQSGANGLPKHSYWLDLWLFIFFDVVVFLFVYFLP) contains a hydrophobic C-terminal region, suggesting potential membrane interaction. To investigate this possibility:

• Membrane topology prediction:

  • Computational algorithms (e.g., TMHMM, MEMSAT) should be applied to predict transmembrane regions

  • The C-terminal sequence (WLFIFFDVVVFLFVYFLP) shows characteristics of a membrane-spanning domain

• Experimental membrane association studies:

  • Cellular fractionation to determine membrane localization

  • Membrane extraction assays using different detergents and buffers

  • Fluorescence microscopy with tagged protein to visualize membrane localization

• Lipid interaction studies:

  • Liposome binding assays

  • Protein-lipid overlay assays to identify specific lipid interactions

  • Fluorescence resonance energy transfer (FRET) to study membrane proximity

• Structural studies in membrane mimetics:

  • NMR studies in micelles or bicelles

  • Molecular dynamics simulations of protein-membrane interactions

Understanding the potential membrane association of this protein would provide significant insight into its cellular function and biological role.

How can differential proteomics approaches be designed to identify pathways affected by this protein?

To identify pathways affected by the Pongo abelii C4orf3 homolog protein, researchers can design differential proteomics experiments similar to those described for related proteins :

• Experimental design:

  • Generate cellular models with knockdown/knockout or overexpression of the C4orf3 homolog

  • Prepare biological replicates (minimum n=3 as used in related studies)

  • Include appropriate controls (e.g., scrambled siRNA, empty vector)

• Sample preparation protocol:

  • Lyse cells in appropriate buffer (e.g., 7M urea, 2M thiourea, 0.1% CHAPS)

  • Sonicate samples (e.g., 80W, ultrasonic 0.2s, intermittent 2s, total 60s)

  • Centrifuge at 15,000×g for 20 min at 4°C to remove debris

  • Determine protein concentration using Bradford assay

• Quantitative proteomics methods:

  • iTRAQ labeling for multiplexed quantitation

  • LC-MS/MS analysis on a high-resolution mass spectrometer

  • Data analysis using appropriate software (e.g., Proteome Discoverer)

  • Statistical criteria: fold changes ≥1.2 or ≤0.83 with p≤0.05

• Pathway analysis:

  • Gene Ontology (GO) enrichment analysis

  • KEGG pathway mapping

  • Protein-protein interaction network analysis

  • Validation of key nodes using targeted proteomics or biochemical assays

This approach would allow identification of protein networks and pathways that are perturbed upon manipulation of C4orf3 homolog expression, providing functional insights.

What approaches can determine if this protein participates in the ESCRT (Endosomal Sorting Complex Required for Transport) pathway?

Given the potential relationship to VPS28 (a known ESCRT-I component) mentioned in the research literature , investigating whether the C4orf3 homolog participates in the ESCRT pathway is a valid research direction:

• Co-localization studies:

  • Immunofluorescence microscopy with markers for endosomal compartments

  • Co-staining with established ESCRT components (VPS28, TSG101, CHMP proteins)

  • Live-cell imaging to track protein dynamics during endosomal sorting

• Protein interaction studies:

  • Co-immunoprecipitation with known ESCRT components

  • Proximity labeling techniques (BioID, APEX) to identify nearby proteins in cellular context

  • Yeast two-hybrid screening against ESCRT library

• Functional assays:

  • EGFR degradation assay (canonical test for ESCRT function)

  • HIV budding assays (ESCRT-dependent process)

  • MVB formation analysis by electron microscopy

  • Cargo sorting assays using fluorescently labeled endocytic cargo

• Genetic interaction studies:

  • Double knockdown/knockout with known ESCRT components

  • Epistasis analysis to position the protein within the pathway

  • Rescue experiments with ESCRT components

This systematic approach would determine whether the C4orf3 homolog functions within the ESCRT pathway and elucidate its specific role in endosomal sorting processes.

How does the C4orf3 homolog from Pongo abelii compare to its human counterpart?

A comparative analysis between the Pongo abelii C4orf3 homolog and its human counterpart would provide evolutionary insights:

• Sequence comparison:

  • Perform pairwise alignment to calculate percent identity and similarity

  • Identify conserved residues that may be functionally important

  • Map species-specific variations that might relate to functional differences

• Expression pattern comparison:

  • Analyze tissue-specific expression profiles in both species

  • Compare developmental expression patterns

  • Identify differentially regulated conditions

• Structural comparison:

  • Generate homology models for both proteins

  • Identify structural conservation and divergence

  • Analyze potential differences in interaction surfaces

• Functional equivalence testing:

  • Express the orangutan protein in human cell lines with CRISPR knockout of the endogenous human gene

  • Assess functional complementation

  • Identify species-specific interaction partners

This comparative approach would help determine whether the function of this protein is conserved between humans and orangutans or has developed species-specific adaptations.

What experimental design would effectively study post-translational modifications of this protein?

To comprehensively characterize post-translational modifications (PTMs) of the Pongo abelii C4orf3 homolog, researchers should implement a multi-faceted mass spectrometry-based workflow:

• Sample preparation strategies:

  • Enrichment techniques for specific PTMs (e.g., phosphopeptide enrichment using TiO₂, ubiquitinated peptide enrichment using anti-K-ε-GG antibodies)

  • Multiple protease digestions (beyond trypsin) to improve sequence coverage

  • PTM-preserving lysis conditions (phosphatase inhibitors, deubiquitinase inhibitors)

• Mass spectrometry approach:

  • High-resolution MS/MS analysis with electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation

  • Data-dependent acquisition for discovery

  • Parallel reaction monitoring for targeted validation

  • Software analysis using tools capable of PTM assignment (similar to Proteome Discoverer used in related studies)

• Site-specific validation:

  • Site-directed mutagenesis of identified PTM sites

  • Functional assays comparing wild-type and mutant forms

  • Generation of site-specific antibodies for key modifications

• Dynamic PTM profiling:

  • Time-course experiments following stimulation

  • Quantitative proteomics to measure changes in modification stoichiometry

  • Inhibitor studies to identify responsible enzymes

This approach would provide a comprehensive map of PTMs on the C4orf3 homolog and insight into their functional significance.