Recombinant Rat UPF0458 protein C7orf42 homolog

What is Rat UPF0458 protein C7orf42 homolog and what are its key characteristics?

Rat UPF0458 protein C7orf42 homolog (UniProt ID: Q6AY76) is a transmembrane protein also known as TMEM248 (Transmembrane protein 248). It is a full-length protein consisting of 314 amino acids that functions as a transmembrane protein with currently uncharacterized specific functions . The protein contains multiple transmembrane domains as suggested by its amino acid sequence, which includes hydrophobic regions characteristic of membrane-spanning segments . When produced recombinantly with an N-terminal His tag, the protein is typically expressed in E. coli expression systems, resulting in a purified product with greater than 90% purity as determined by SDS-PAGE analysis .

How does Rat UPF0458 protein C7orf42 homolog compare to its bovine counterpart?

Comparative analysis of rat and bovine UPF0458 protein C7orf42 homologs reveals high sequence conservation with subtle species-specific variations:

The high degree of sequence conservation suggests evolutionary preservation of critical functional domains, while variations may reflect species-specific adaptations or neutral mutations . These differences may impact protein-protein interactions or functional properties that should be considered when extrapolating findings between species.

What are the optimal expression systems for producing recombinant Rat UPF0458 protein?

For recombinant production of Rat UPF0458 protein C7orf42 homolog, E. coli expression systems have been successfully employed as documented in commercial production protocols . When designing expression experiments, researchers should consider:

• Vector selection: Vectors containing strong promoters (T7, tac) with N-terminal His-tag coding sequences optimize purification efficiency.

• Expression conditions: Typical induction protocols involve IPTG induction at OD600 0.6-0.8, with expression at reduced temperatures (16-25°C) to enhance proper folding of transmembrane regions.

• Purification strategy: Immobilized metal affinity chromatography (IMAC) utilizing the His-tag, followed by size exclusion chromatography produces high purity protein (>90% as determined by SDS-PAGE) .

• Alternative systems: For studies requiring post-translational modifications or membrane insertion studies, mammalian or insect cell expression systems may be preferable, though these have not been extensively documented for this specific protein.

What are the proper storage and reconstitution protocols for recombinant Rat UPF0458 protein?

Proper handling of recombinant Rat UPF0458 protein C7orf42 homolog is critical to maintain its structural integrity and biological activity. Based on established protocols:

Storage recommendations:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots may be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended: 50%)

• Aliquot for long-term storage at -20°C/-80°C

The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which has been optimized to maintain protein stability . Researchers should avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of biological activity.

How can researchers validate the structural integrity of recombinant Rat UPF0458 protein?

Validation of recombinant Rat UPF0458 protein C7orf42 homolog should employ multiple complementary approaches:

• SDS-PAGE analysis: Confirms protein size (expected MW based on sequence) and purity (>90% is standard for most applications)

• Western blotting: Using anti-His tag antibodies to confirm presence of intact N-terminal tag

• Mass spectrometry: For precise mass determination and sequence coverage analysis

• Circular dichroism (CD): To assess secondary structure elements and proper folding

• Thermal shift assays: To evaluate protein stability under different buffer conditions

• Functional assays: Though specific activities are not well-characterized for this protein, membrane integration or protein-protein interaction assays may be developed based on hypothesized functions

Researchers should document batch-to-batch variation by maintaining consistent quality control metrics across preparations, especially when conducting long-term studies requiring multiple protein preparations.

What approaches can be used to investigate the cellular localization of Rat UPF0458/TMEM248?

Investigating the cellular localization of Rat UPF0458/TMEM248 requires multiple complementary techniques:

• Immunofluorescence microscopy: Using validated antibodies against Rat UPF0458/TMEM248 or tagged recombinant versions. Based on its predicted transmembrane domains, co-localization studies with markers for cellular compartments (including plasma membrane, ER, Golgi) should be conducted.

• Subcellular fractionation: Differential centrifugation followed by Western blotting of fractions can biochemically confirm the protein's compartmentalization.

• Live-cell imaging: Expression of fluorescently-tagged UPF0458/TMEM248 constructs allows real-time visualization of localization and dynamics.

• Electron microscopy: Immunogold labeling can provide high-resolution localization within membrane structures.

The amino acid sequence of Rat UPF0458 contains hydrophobic regions consistent with transmembrane domains (e.g., "VFMISVSAMAIAFLTLGYFFKI"), suggesting plasma membrane localization, but experimental verification is essential . Comparative analysis with data from homologs in other species can provide additional insights into conserved localization patterns.

How might researchers investigate potential functions of Rat UPF0458/TMEM248 in cellular systems?

While the specific functions of Rat UPF0458/TMEM248 remain largely uncharacterized, several experimental approaches can help elucidate its biological roles:

• Gene silencing/knockout studies: Using siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate expression, followed by phenotypic analysis.

• Overexpression studies: Expressing recombinant protein to identify gain-of-function phenotypes.

• Protein-protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Proximity labeling (BioID, APEX)

  • Split-reporter assays for membrane protein interactions

• Comparative genomics: Analysis of conserved domains across species may provide functional clues. The high sequence conservation between rat and bovine homologs (particularly in C-terminal regions) suggests functionally important domains .

• Structure-function analysis: Creating targeted mutations in conserved residues to identify domains critical for function.

Research could examine potential involvement in transmembrane signaling, molecular transport, or structural roles in membrane organization based on its predicted transmembrane topology.

What bioinformatic approaches can help predict functional domains in Rat UPF0458/TMEM248?

Computational analysis of Rat UPF0458/TMEM248 can provide valuable insights into its potential functional domains and evolutionary relationships:

• Transmembrane domain prediction: Tools like TMHMM, Phobius, or TOPCONS can identify potential membrane-spanning regions in the sequence "MFSINPLENLKLYISSRPPLVVFMISVSAMAIAFLTLGYFFKIKEIKSPEMAEDWNTFLL..."

• Conserved domain analysis: InterPro, PFAM, or CDD searches can identify known functional domains.

• Secondary structure prediction: PSIPRED or JPred can predict alpha-helices, beta-sheets, and unstructured regions.

• Post-translational modification sites: NetPhos, NetOGlyc, NetNGlyc for potential phosphorylation and glycosylation sites.

• Homology modeling: Using related proteins with known structures as templates.

• Evolutionary conservation analysis: ConSurf or similar tools to identify residues under evolutionary constraint, which often correlate with functional importance.

• Protein-protein interaction prediction: STRING database or PRISM for potential interaction partners.

A multi-faceted bioinformatic approach should be combined with experimental validation to develop testable hypotheses about UPF0458/TMEM248 function.

What are the challenges in purifying and studying transmembrane proteins like UPF0458/TMEM248?

Working with transmembrane proteins like Rat UPF0458/TMEM248 presents several technical challenges that researchers should anticipate:

• Expression and solubility issues:

  • Membrane proteins often form inclusion bodies in bacterial systems

  • Toxicity to host cells when overexpressed

  • Lower yields compared to soluble proteins

• Purification complications:

  • Requirement for detergents or amphipols to maintain solubility

  • Potential for aggregation during purification

  • Difficulty in removing all detergent without protein precipitation

• Structural integrity concerns:

  • Native conformation may depend on membrane environment

  • Detergents may not fully mimic the lipid bilayer

  • Potential for misfolding outside the cellular membrane context

• Functional assessment limitations:

  • Biochemical assays may not reflect in vivo activity

  • Reconstitution into artificial membranes may be necessary

  • Difficulty in establishing physiologically relevant assays

When working with recombinant Rat UPF0458 protein, researchers should carefully optimize buffer conditions, consider membrane-mimetic systems (nanodiscs, liposomes), and validate that the purified protein retains native-like properties .

How can researchers develop specific antibodies against Rat UPF0458/TMEM248?

Developing specific antibodies against Rat UPF0458/TMEM248 requires careful epitope selection and validation strategies:

• Epitope selection approaches:

  • Target extracellular or cytoplasmic domains rather than transmembrane regions

  • Use hydrophilic, surface-exposed regions predicted by structural analysis

  • Consider unique regions that differ from homologs to enhance specificity

  • The amino acid sequence provided in the product details can guide epitope selection

• Immunization strategies:

  • Use of synthetic peptides corresponding to selected epitopes

  • Recombinant protein fragments expressed in E. coli

  • DNA immunization encoding antigenic regions

• Validation methods:

  • Western blotting against recombinant protein and tissue lysates

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with appropriate controls

  • Testing in tissues from knockout animals if available

  • Cross-reactivity testing against homologs from other species

• Monoclonal vs. polyclonal considerations:

  • Monoclonal antibodies offer higher specificity but limited epitope recognition

  • Polyclonal antibodies provide broader epitope recognition but potential cross-reactivity

Researchers should document all validation steps and clearly specify the recognized epitopes when reporting antibody-based experiments.

What controls should be included when studying the effects of recombinant Rat UPF0458 protein in experimental systems?

Rigorous experimental design for studies involving recombinant Rat UPF0458 protein C7orf42 homolog should include these essential controls:

• Negative controls:

  • Buffer-only treatments matching the reconstitution buffer (Tris/PBS-based buffer with 6% Trehalose, pH 8.0)

  • Irrelevant proteins of similar size with matching tags

  • Heat-denatured UPF0458 protein to confirm activity requires native conformation

• Positive controls:

  • Known bioactive proteins in the same experimental system

  • Established inducers of the cellular responses being measured

• Tag controls:

  • Proteins with identical tags but different sequences

  • If possible, comparing tagged vs. untagged versions to assess tag interference

• Dose-response relationships:

  • Testing multiple concentrations to establish biological relevance

  • Determining EC50/IC50 values where appropriate

• Timing controls:

  • Time-course experiments to establish optimal treatment duration

  • Reversibility studies by protein washout

• Species-specificity controls:

  • Comparing effects of rat vs. bovine homologs to assess conservation of function

  • The amino acid sequence comparison shows both conserved and variable regions that may affect function

• Blocking controls:

  • Specific antibodies or competing ligands to confirm specificity of observed effects

Proper documentation of batch information, including purity assessment and storage conditions, is essential for reproducibility across experiments.

What approaches can be used to compare Rat UPF0458/TMEM248 with homologs from other species?

Comparative analysis of Rat UPF0458/TMEM248 with homologs from different species can provide valuable insights into evolutionary conservation and potential functional domains:

• Sequence alignment methods:

  • Multiple sequence alignment using CLUSTAL, MUSCLE, or T-Coffee

  • Construction of phylogenetic trees to visualize evolutionary relationships

  • Calculation of percent identity and similarity between homologs

• Structure-based comparisons:

  • Homology modeling based on related structures if available

  • Prediction of conserved structural elements across species

  • Analysis of conservation in transmembrane domains versus loop regions

• Functional domain analysis:

  • Identification of conserved motifs potentially related to function

  • Comparison of the rat sequence (MFSINPLENLKLYISSRPPL...) with bovine homolog (MFNINPLENLKLYISSRPPL...)

  • Mapping of conserved regions to predicted functional domains

• Evolutionary rate analysis:

  • Calculation of Ka/Ks ratios to identify regions under purifying or positive selection

  • Identification of species-specific variations that might relate to functional differences

• Expression pattern comparison:

  • Analysis of tissue-specific expression across species

  • Examination of developmental regulation patterns

Based on the available sequence data, rat and bovine UPF0458 homologs share high sequence identity, particularly in the C-terminal region, suggesting evolutionary conservation of critical functional domains .

How should researchers interpret unexpected results in UPF0458/TMEM248 functional studies?

When encountering unexpected results in studies involving Rat UPF0458/TMEM248, researchers should systematically evaluate multiple explanations:

• Technical considerations:

  • Protein quality issues (degradation, aggregation, improper folding)

  • Assay limitations or artifacts

  • Interference from tags or fusion partners

  • Variations in reconstitution procedures

• Biological complexity factors:

  • Context-dependent protein functions

  • Concentration-dependent effects (physiological vs. supraphysiological)

  • Cell type-specific responses

  • Species-specific differences when comparing to homologs

• Interpretation frameworks:

  • Consider pleiotropic functions of the protein

  • Evaluate potential novel functions not previously described

  • Examine convergence with functions of related proteins

  • Assess consistency with evolutionary conservation patterns

• Validation approaches:

  • Reproduce findings using alternative methodologies

  • Test in multiple cell types or model systems

  • Use complementary gain-of-function and loss-of-function approaches

  • Compare with homologs from other species (e.g., bovine UPF0458)

When reporting unexpected findings, researchers should comprehensively document experimental conditions, include all relevant controls, and acknowledge limitations in current understanding of UPF0458/TMEM248 function.

What bioinformatic resources are most valuable for researching UPF0458/TMEM248 function?

Researchers studying Rat UPF0458/TMEM248 can leverage numerous bioinformatic resources:

• Primary databases:

  • UniProt (Q6AY76 for rat, Q2YDM0 for bovine homolog)

  • NCBI Gene (human homolog: TMEM248)

  • Ensembl Genome Browser

• Structural prediction tools:

  • AlphaFold for protein structure prediction

  • TMHMM, TOPCONS for transmembrane domain prediction

  • NetSurfP for surface accessibility

  • PSIPRED for secondary structure prediction

• Functional annotation resources:

  • Gene Ontology (GO) annotations

  • KEGG for pathway involvement

  • STRING for protein-protein interaction networks

  • Pfam for protein domain identification

• Expression databases:

  • Gene Expression Omnibus (GEO)

  • Expression Atlas

  • GTEx for tissue-specific expression patterns

• Disease association resources:

  • Open Targets Platform

  • DisGeNET

  • GWAS Catalog

• Comparative genomics tools:

  • OrthoDB for ortholog identification

  • Ensembl Compara for evolutionary comparisons

  • VISTA for genomic conservation analysis

These resources should be used in an integrated manner to develop comprehensive hypotheses about UPF0458/TMEM248 function that can be experimentally tested.

What are emerging techniques that could advance understanding of UPF0458/TMEM248 function?

Several cutting-edge methodologies could significantly enhance our understanding of Rat UPF0458/TMEM248:

• Cryo-electron microscopy: For high-resolution structural determination of this membrane protein, potentially revealing functional domains and interaction surfaces.

• Single-cell technologies: Examining expression patterns and variability at single-cell resolution across tissues and developmental stages.

• Proximity labeling techniques: BioID or APEX2 fusion proteins to identify the protein's interactome in living cells, particularly valuable for membrane proteins.

• Organoid models: Studying the protein's function in three-dimensional tissue models that better recapitulate in vivo environments.

• CRISPR-based genetic screens: Identifying genetic interactions and pathways connected to UPF0458/TMEM248 function.

• Integrative multi-omics approaches: Combining transcriptomics, proteomics, and metabolomics to comprehensively assess the impact of UPF0458/TMEM248 perturbation.

• Advanced imaging techniques: Super-resolution microscopy and correlative light-electron microscopy for precise subcellular localization studies.

• Protein engineering approaches: Creating chimeric proteins between rat and other species homologs to identify functionally important domains.

Given the transmembrane nature of UPF0458/TMEM248, technologies specifically designed for membrane protein analysis will be particularly valuable for future research .

How could knowledge from UPF0458/TMEM248 research potentially apply to human health?

While direct clinical applications remain speculative given the limited characterization of UPF0458/TMEM248, several potential translational pathways merit investigation:

• Comparative studies with human TMEM248: The human homolog of this protein could have relevant functions in health and disease. Research on the rat protein may provide valuable insights applicable to human biology .

• Inflammatory pathway interactions: Recent research on hybrid proteins has identified regulatory roles in inflammatory pathways relevant to conditions like colitis. Similarly, TMEM248 might participate in immune-related signaling pathways .

• Membrane protein therapeutic targets: As a transmembrane protein, UPF0458/TMEM248 belongs to a class of proteins that represents approximately 60% of current drug targets. Better understanding of this protein could potentially reveal new therapeutic approaches.

• Biomarker potential: If expression patterns correlate with specific pathological states, UPF0458/TMEM248 could potentially serve as a biomarker.

• Model system applications: The recombinant protein could serve as a tool for screening compounds that modulate membrane protein function or stability, with potential applications in drug discovery pipelines.