Recombinant Mouse UPF0575 protein C19orf67 homolog

What is UPF0575 protein C19orf67 homolog in mice?

UPF0575 protein C19orf67 homolog is a protein encoded by the 1700067K01Rik gene in Mus musculus (house mouse) . It is the murine ortholog of the human C19orf67 gene product. The "UPF" designation indicates it belongs to an uncharacterized protein family, suggesting its function has not been fully elucidated. The protein contains the evolutionarily conserved DUF3314 domain that is predicted to form numerous alpha helices, potentially essential to its function . Based on homology studies, this protein is conserved across mammals and is found in some reptiles and most jawed fish, indicating potential evolutionary importance .

What are the key structural characteristics of mouse UPF0575 protein C19orf67 homolog?

While specific mouse protein data is limited in the search results, we can extrapolate from human ortholog data that the mouse protein likely shares similar characteristics. The human ortholog contains 358 amino acids with a predicted molecular weight of approximately 40kDa, a charge of -11, and an isoelectric point of 5 . A notable feature is its high proline content (12.3%), which exceeds that of 95% of comparable human proteins . The protein contains the highly conserved DUF3314 region which is predicted to form numerous alpha helices . Recombinant versions of this protein are typically produced in E. coli expression systems .

Where is UPF0575 protein C19orf67 homolog typically expressed in mice?

Based on information from the human ortholog, we can infer that the mouse UPF0575 protein C19orf67 homolog is likely expressed at low levels throughout the body with potentially higher expression in specific tissues such as testis and breast tissue (as observed in humans) . Researchers should conduct tissue-specific expression analyses in mice to confirm these patterns, potentially using techniques such as qRT-PCR, RNA-seq, or immunohistochemistry with specific antibodies against the mouse protein to determine its precise expression pattern across different tissues and developmental stages.

What are the recommended methods for studying the function of mouse UPF0575 protein C19orf67 homolog?

To elucidate the function of this relatively understudied protein, researchers should consider a multi-faceted approach:

• Protein interaction studies: Employ techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity labeling methods (BioID or APEX) to identify protein interaction partners.

• Subcellular localization experiments: Based on the prediction that the human ortholog localizes to the nucleus , researchers should verify the subcellular localization in mouse cells using fluorescently tagged versions of the protein or immunocytochemistry with specific antibodies.

• Gene knockout or knockdown studies: Generate knockout mice using CRISPR-Cas9 or employ siRNA/shRNA approaches in cell culture to observe phenotypic changes.

• Proteomic analyses: Utilize mass spectrometry-based approaches to identify post-translational modifications that might regulate protein function.

• Transcriptomic analyses: Conduct RNA-seq after manipulation of protein expression to identify downstream effectors or regulatory pathways.

These methodologies should be combined with careful phenotypic characterization to correlate molecular changes with physiological effects.

How can I design experiments to investigate the role of the DUF3314 domain in mouse UPF0575 protein C19orf67 homolog?

The DUF3314 domain is highly conserved and predicted to form numerous alpha helices, suggesting functional importance . To investigate its role:

• Structure-function analysis: Create a series of deletion mutants or point mutations within the DUF3314 domain and assess their effects on protein function, localization, and interaction partners.

• Domain swapping experiments: Replace the DUF3314 domain with homologous domains from other species to assess functional conservation.

• Structural studies: Express and purify the DUF3314 domain for X-ray crystallography, NMR spectroscopy, or cryo-EM analysis to determine its three-dimensional structure.

• Molecular dynamics simulations: Use computational approaches to predict conformational changes of the domain under different conditions.

• Evolutionary analysis: Compare the DUF3314 sequences across species to identify highly conserved residues that may be essential for function.

Each approach should be followed by functional validation to connect structural features with biological activity.

What expression systems are optimal for producing recombinant mouse UPF0575 protein C19orf67 homolog?

The choice should be guided by the intended experimental use, required protein purity, and whether post-translational modifications are essential for the protein's function.

What purification strategies are most effective for recombinant mouse UPF0575 protein C19orf67 homolog?

While specific purification protocols for this protein are not detailed in the search results, effective strategies can be designed based on general protein characteristics:

• Affinity chromatography: Use of histidine tags (as seen in commercial products ) enables purification via nickel or cobalt affinity columns. Based on available recombinant products, a C-terminal His tag approach may be effective .

• Ion exchange chromatography: Given the predicted isoelectric point of 5 (based on human ortholog data ), cation exchange chromatography at pH below 5 or anion exchange above pH 5 could be effective.

• Size exclusion chromatography: As a polishing step to separate the target protein from aggregates or degradation products.

• Optimization considerations:

  • Buffer conditions: Test various pH values and salt concentrations to maximize protein stability and solubility

  • Protease inhibitors: Include during extraction and initial purification steps

  • Reducing agents: Consider including DTT or β-mercaptoethanol if the protein contains cysteine residues

  • Temperature: Perform purification at 4°C to minimize degradation

A typical purification workflow might include initial capture by affinity chromatography, followed by ion exchange and size exclusion as polishing steps.

How conserved is UPF0575 protein C19orf67 homolog across species, and what does this suggest about its function?

UPF0575 protein C19orf67 homolog shows significant evolutionary conservation. Orthologs have been identified in many mammals, some reptiles, and most jawed fish . This broad conservation suggests an important biological function that has been maintained throughout vertebrate evolution.

The protein contains a highly conserved DUF3314 domain that appears to be critical to its function . The conservation pattern of this domain can provide clues about functionally important residues or regions within the protein.

Conservation data across selected species:

Researchers can use phylogenetic analyses and sequence alignments to identify highly conserved motifs or residues within the protein, which often correspond to functionally critical regions. Molecular evolution studies, such as dN/dS ratio analysis, can also reveal whether certain regions of the protein are under purifying or positive selection, providing additional insights into functional constraints.

How do the different isoforms of mouse UPF0575 protein C19orf67 homolog compare with those in other species?

Based on the search results, multiple isoforms of UPF0575 protein C19orf67 homolog have been identified in various species. For example, in Papio anubis (olive baboon), at least two isoforms (X1 and X2) have been documented . The human ortholog appears to have three transcript variants, although only two are protein-coding .

For comprehensive isoform analysis, researchers should:

• Conduct detailed RNA-seq analyses across different tissues and developmental stages to identify all potential mouse isoforms

• Perform comparative analyses of isoform expression patterns across species

• Investigate whether different isoforms have distinct subcellular localizations or interaction partners

• Determine if isoform ratios change under different physiological or pathological conditions

Understanding isoform diversity can provide insights into potential tissue-specific or context-dependent functions of this protein family.

What factors regulate the expression of mouse UPF0575 protein C19orf67 homolog?

While specific regulatory mechanisms for mouse UPF0575 protein C19orf67 homolog are not detailed in the search results, researchers interested in understanding its regulation should investigate:

• Transcriptional regulation: Analyze the promoter region to identify potential transcription factor binding sites. Conduct chromatin immunoprecipitation (ChIP) experiments to confirm these interactions.

• Epigenetic regulation: Examine DNA methylation patterns and histone modifications in the gene's regulatory regions across different tissues or developmental stages.

• Post-transcriptional regulation: Investigate potential microRNA binding sites in the mRNA that might regulate translation efficiency or mRNA stability.

• Post-translational modifications: Identify potential phosphorylation, ubiquitination, or other modification sites that might regulate protein stability or activity.

• Signaling pathways: Determine if expression changes in response to specific signaling pathways or cellular stresses.

Experiments should include reporter assays with the gene's promoter, RNA stability assessments, and protein half-life determinations to comprehensively understand its regulation.

How can I effectively measure mouse UPF0575 protein C19orf67 homolog expression in different experimental contexts?

To accurately measure expression levels of mouse UPF0575 protein C19orf67 homolog:

• mRNA quantification:

  • qRT-PCR: Design specific primers spanning exon-exon junctions to avoid genomic DNA amplification

  • RNA-seq: For genome-wide expression analysis and detection of potential novel isoforms

  • Northern blotting: For visualization of transcript size and abundance

• Protein quantification:

  • Western blotting: Using specific antibodies against the mouse protein

  • ELISA: For quantitative measurement in tissue lysates or biological fluids

  • Mass spectrometry: For absolute quantification and identification of post-translational modifications

• Spatial expression:

  • In situ hybridization: To visualize mRNA distribution in tissues

  • Immunohistochemistry: To detect protein localization in tissue sections

  • Single-cell RNA-seq: To analyze expression heterogeneity within tissues

• Experimental considerations:

  • Include appropriate housekeeping genes or proteins as internal controls

  • Validate antibody specificity using knockout or knockdown controls

  • Consider temporal dynamics of expression when designing experiments

These methodologies should be selected based on the specific research question, required sensitivity, and available resources.

What phenotypes are associated with altered expression of mouse UPF0575 protein C19orf67 homolog?

Since UPF0575 protein C19orf67 homolog is relatively understudied, specific phenotypes associated with its altered expression are not well documented in the search results. Researchers investigating phenotypic effects should:

• Generate knockout or knockdown models (whole organism or tissue-specific) using CRISPR-Cas9 or RNA interference technologies.

• Create overexpression models to assess gain-of-function effects.

• Conduct comprehensive phenotypic analyses including:

  • Developmental assessments: Embryonic viability, growth patterns, organ development

  • Histological examinations: Tissue architecture and cellular morphology

  • Behavioral studies: For potential neurological phenotypes

  • Reproductive assessments: Given potential testis expression based on human data

  • Molecular profiling: Transcriptomics and proteomics to identify affected pathways

• Investigate cell-autonomous effects in primary cells isolated from modified animals or in cell lines with manipulated expression.

These approaches should be complemented with rescue experiments to confirm the specificity of observed phenotypes.

How does mouse UPF0575 protein C19orf67 homolog interact with other cellular proteins and pathways?

To elucidate the interaction network and pathway involvement of mouse UPF0575 protein C19orf67 homolog:

• Protein interaction mapping:

  • Immunoprecipitation followed by mass spectrometry (IP-MS)

  • Yeast two-hybrid screening

  • Proximity-dependent biotin identification (BioID)

  • Cross-linking mass spectrometry to capture transient interactions

• Pathway analysis:

  • Phosphoproteomic analysis after protein depletion to identify affected signaling pathways

  • Transcriptomic profiling to identify genes whose expression changes upon protein manipulation

  • Genetic interaction screens to identify synthetic lethal or synthetic viable interactions

• Co-localization studies:

  • Fluorescence co-localization with potential interacting partners

  • FRET or BRET analyses to confirm direct interactions in living cells

• Functional validation:

  • Mutagenesis of key residues to disrupt specific interactions

  • Domain deletion analysis to map interaction interfaces

  • Competitive peptide inhibition to confirm specificity of interactions

These approaches should be integrated to build a comprehensive understanding of how this protein functions within broader cellular networks.