Recombinant Pongo abelii UPF0542 protein C5orf43 homolog

What is the UPF0542 protein C5orf43 homolog and how is it classified?

The UPF0542 protein C5orf43 homolog is a protein originally identified in Pongo abelii (Sumatran orangutan) that shares sequence homology with the human C5orf43 protein. It belongs to the UPF (Uncharacterized Protein Family) classification, specifically UPF0542, indicating that its function has not been fully characterized. This 74-amino acid protein has the UniProt accession number Q5R4D8 and represents a full-length expression of the gene region 1-74 .

What are the optimal storage conditions for this recombinant protein?

The recombinant UPF0542 protein C5orf43 homolog should be stored at -20°C for regular use, and at -80°C for extended storage. The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for this specific protein. Researchers should avoid repeated freeze-thaw cycles as these can compromise protein integrity. For short-term work, storing working aliquots at 4°C for up to one week is recommended to minimize degradation while maintaining accessibility .

What expression systems are most effective for producing this protein?

Based on comparable recombinant proteins from Pongo abelii, both yeast and E. coli expression systems can be utilized for heterologous expression. The choice of expression system should depend on the specific research requirements:

When using either system, codon optimization for the host organism should be considered to enhance expression efficiency .

How should researchers approach experimental design when studying uncharacterized proteins like UPF0542?

A systematic approach is recommended:

• Sequence analysis: Conduct comprehensive bioinformatic analysis to identify conserved domains, motifs, and potential functional sites

• Expression profiling: Determine tissue distribution and expression patterns across developmental stages

• Subcellular localization: Use fluorescent tagging (GFP fusion) to establish cellular compartment localization

• Interaction studies: Employ co-immunoprecipitation, yeast two-hybrid, or proximity labeling techniques to identify binding partners

• Loss/gain-of-function studies: Use CRISPR-Cas9 or RNAi methods to assess phenotypic effects

• Comparative functional assays: Test hypothesized functions based on structurally similar proteins

This systematic pipeline allows for iterative hypothesis refinement about the protein's function .

What approaches can researchers use to determine potential functions of this uncharacterized protein?

Since the UPF0542 protein C5orf43 homolog belongs to an uncharacterized protein family, researchers can employ multiple complementary strategies:

• Homology-based prediction: Compare with functionally characterized homologs across species

• Domain recognition: Identify functional domains that might suggest biochemical activity

• Interactome mapping: Identify binding partners to place the protein in functional networks

• Structural biology: Determine 3D structure through X-ray crystallography or cryo-EM

• Phenotypic screening: Observe cellular changes upon protein overexpression or knockdown

• Transcriptomics/proteomics: Analyze global changes in expression profiles when modulating the protein

Integrating data from these approaches can provide convergent evidence for functional hypotheses .

How can researchers effectively study protein-protein interactions involving UPF0542?

For an uncharacterized protein like UPF0542, a multi-method approach is recommended:

• Affinity purification coupled with mass spectrometry (AP-MS) to identify stable interaction partners

• Proximity-dependent biotin identification (BioID) to capture transient interactions

• Split-reporter assays (such as yeast two-hybrid or split-luciferase) to validate direct interactions

• Co-localization studies using fluorescence microscopy to confirm spatial relationships

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding kinetics and affinity

Researchers should consider both in vitro and cellular context interactions, as the behavior of membrane-associated proteins often depends on their lipid environment .

What techniques can help determine if UPF0542 has enzymatic activity?

To investigate potential enzymatic functions:

• Substrate screening: Test activity against classes of substrates based on homology predictions

• Activity-based protein profiling: Use chemical probes that react with active site residues

• Metabolomic profiling: Compare metabolite changes in cells with normal versus altered protein levels

• Structural analysis: Identify potential catalytic residues and substrate-binding pockets

• Enzyme kinetics: If activity is identified, characterize kinetic parameters (Km, Vmax, kcat)

The presence of conserved amino acid motifs in the sequence may provide hints about potential catalytic activities that should be prioritized for testing .

How does Pongo abelii UPF0542 compare to its human counterpart?

Detailed sequence alignment and structural comparison between the Pongo abelii UPF0542 protein and its human counterpart would provide valuable insights into evolutionary conservation. While specific comparison data is not directly available in the search results, researchers should analyze:

• Sequence identity and similarity percentages

• Conservation of key functional domains

• Patterns of selective pressure (Ka/Ks ratios)

• Difference in expression patterns between species

• Potential species-specific post-translational modifications

This comparative analysis can highlight functionally important regions that have been conserved throughout primate evolution .

What can phylogenetic analysis tell us about the evolutionary history of UPF0542?

Phylogenetic analysis of UPF0542 across multiple species can reveal:

• The evolutionary age of this protein family

• Patterns of gene duplication and divergence

• Potential neofunctionalization or subfunctionalization events

• Correlation between protein evolution and species adaptation

• Identification of taxonomic groups where the protein has undergone accelerated evolution

Researchers should construct phylogenetic trees using maximum likelihood or Bayesian methods and analyze patterns of sequence conservation across evolutionary time .

Are there functional differences between UPF0542 and other UPF protein family members in Pongo abelii?

When comparing UPF0542 to other UPF proteins found in Pongo abelii (such as UPF0554, UPF0489):

• Sequence similarity analysis may reveal shared domains despite different chromosomal origins

• Subcellular localization studies can identify distinct or overlapping cellular compartmentalization

• Expression profiling across tissues might show tissue-specific functions

• Interaction network mapping could reveal unique or redundant cellular pathways

The comparison with UPF0554 protein C2orf43 homolog and UPF0489 protein C5orf22 homolog could provide valuable insights into functional diversification within this protein superfamily .

What are common challenges when working with recombinant membrane-associated proteins?

If UPF0542 is indeed membrane-associated as its sequence suggests, researchers might encounter:

• Solubility issues during purification requiring specialized detergents

• Proper folding challenges in heterologous expression systems

• Maintenance of native conformation during experimental manipulation

• Difficulties in crystallization for structural studies

• Potential for aggregation during concentration or storage

To address these challenges, researchers should consider:

• Using mild non-ionic detergents for extraction

• Employing lipid nanodiscs or liposomes to maintain native environment

• Testing multiple buffer conditions for optimal stability

• Using size exclusion chromatography to monitor oligomeric state

• Considering membrane mimetics for structural studies .

How can researchers validate antibody specificity for UPF0542 protein studies?

When developing or selecting antibodies against UPF0542:

• Perform Western blot using both recombinant protein and endogenous samples

• Include knockout/knockdown controls to verify specificity

• Test cross-reactivity with related proteins, especially other UPF family members

• Validate using orthogonal methods (mass spectrometry, immunoprecipitation)

• Check recognition of native versus denatured protein if conformational epitopes are suspected

The validation approach should be tailored to the intended application (Western blot, immunofluorescence, chromatin immunoprecipitation, etc.) .

What quality control measures ensure optimal protein activity for functional studies?

To ensure experimental reproducibility and reliability:

• Purity assessment: SDS-PAGE and mass spectrometry to confirm >95% purity

• Integrity verification: Western blot to check for degradation products

• Functional validation: Activity assays if function is known or binding assays with known partners

• Structural confirmation: Circular dichroism to verify secondary structure content

• Batch consistency: Lot-to-lot comparison using established quality metrics

The protein is typically supplied as 50 μg per vial, which should be sufficient for multiple experiments, but researchers should establish minimum active concentrations for their specific assays .

How can CRISPR-Cas9 technology be applied to study UPF0542 function?

CRISPR-Cas9 provides powerful approaches for functional characterization:

• Gene knockout: Complete elimination of the protein to observe phenotypic consequences

• Knock-in tags: Introduction of epitope or fluorescent tags at endogenous loci

• Domain deletion/mutation: Targeted modification of specific protein regions

• CRISPRi/CRISPRa: Modulation of expression levels without sequence alteration

• Base editing: Introduction of specific amino acid changes to test functional hypotheses

When designing gRNAs, researchers should consider species-specific genomic context and potential off-target effects .

What high-throughput approaches can accelerate functional discovery for UPF0542?

Modern high-throughput technologies can rapidly generate functional hypotheses:

• Pooled CRISPR screens to identify genetic interactions

• Protein microarrays to assess binding to various ligands or proteins

• Thermal proteome profiling to identify small molecule interactions

• Single-cell transcriptomics after perturbation to reveal pathway involvement

• Spatial proteomics to determine subcellular dynamics and translocation events

These approaches can provide unbiased insights that might not emerge from hypothesis-driven experiments alone .

How might structural biology approaches inform UPF0542 function?

Structural determination can provide critical functional insights:

• X-ray crystallography: Highest resolution but requires protein crystallization

• Cryo-EM: Suitable for larger complexes and membrane proteins

• NMR spectroscopy: Provides dynamic information in solution

• AlphaFold2 or RoseTTAFold: AI-based structure prediction when experimental structures are challenging

• Molecular dynamics simulations: Investigate conformational flexibility and potential binding sites

Structural information can reveal cryptic binding sites, allosteric mechanisms, and potential catalytic centers that inform functional hypotheses .

What is known about the relationship between UPF0542-like proteins and human disorders?

While direct information about UPF0542's role in disease is limited in the search results, researchers investigating this protein should:

• Examine genome-wide association studies (GWAS) for variants in the human homolog

• Investigate tissue expression patterns in disease versus normal conditions

• Consider potential roles in fundamental cellular processes that could impact disease

• Analyze protein interaction networks for connections to known disease-associated proteins

• Look for dysregulation in cancer transcriptome/proteome datasets

Understanding the function of uncharacterized proteins often reveals unexpected connections to human disease mechanisms .

How can studies of UPF0542 inform understanding of protein evolution in primates?

Comparative studies between human and non-human primate proteins can reveal:

• Conservation patterns suggesting functional importance

• Species-specific adaptations that might relate to physiological differences

• Evolutionary rates that correlate with environmental or behavioral changes

• Insights into protein family expansion or contraction across lineages

• Evidence of positive selection that might indicate adaptive functions

This evolutionary context provides valuable perspective on protein function and can inform studies of human biology and disease .