Recombinant Xenopus laevis UPF0458 protein C7orf42 homolog

How is this protein conserved across species?

The UPF0458 protein C7orf42 homolog appears to be evolutionarily conserved across vertebrates. Homologs have been identified in multiple species including bovine (TMEM248/C7orf42/C25H7orf42), rat (Tmem248), human (C7orf42/TMEM248), and zebrafish (zgc:103561/tmem248) . The conservation suggests functional importance, though detailed comparative sequence analysis would be needed to identify critical domains and motifs that may indicate specific functional roles.

What expression systems are most effective for producing recombinant Xenopus laevis UPF0458 protein?

Multiple expression systems have been employed successfully for this protein:

• E. coli expression: The protein can be expressed in E. coli, though it often forms inclusion bodies that require refolding .

• Cell-free expression systems: These provide greater than 85% purity as determined by SDS-PAGE .

• Baculovirus expression systems: Used for higher eukaryotic protein expression with better post-translational modifications .

• Mammalian cell expression: HEK293T cells have been used for expression of Xenopus proteins with proper folding and post-translational modifications .

The choice depends on the experimental requirements. For structural studies requiring large quantities, E. coli expression with subsequent refolding may be suitable. For functional studies, insect or mammalian expression systems that provide proper folding and post-translational modifications may be preferable.

What refolding protocols have been optimized for UPF0458 protein from inclusion bodies?

When expressed in E. coli, the protein typically forms inclusion bodies that require refolding. Two effective refolding buffer compositions have been identified:

• Arginine buffer: 50 mM Tris pH 10.0, 10 mM CaCl₂, 2 mM GSSG, 0.2 mM GSH, and 0.3 M L-arginine

• NDSB-201 buffer: 50 mM Tris pH 10.0, 10 mM CaCl₂, 2 mM GSSG, 0.2 mM GSH, and 0.75 M NDSB-201

The arginine buffer has been preferred for larger-scale production due to higher yield and lower cost compared to NDSB-201 . The refolding process typically involves dissolving inclusion bodies in urea, followed by gradual dilution into the refolding buffer while maintaining appropriate redox conditions for disulfide bond formation.

What purification strategies yield the highest purity of recombinant UPF0458 protein?

Several purification approaches have been employed:

• His-tag affinity chromatography: For His-tagged versions, Ni-NTA affinity chromatography offers high binding capacity and specificity .

• Affinity chromatography: If the protein retains its binding properties, specific ligands (such as those used for XCGL-1) can be immobilized on resins for purification .

• Size exclusion chromatography: Often used as a polishing step to separate monomeric from oligomeric forms and remove aggregates.

Purification typically achieves greater than 90% purity as determined by SDS-PAGE . For highest purity, a combination of affinity chromatography followed by size exclusion is recommended.

What is known about the disulfide bonding pattern in UPF0458 protein?

While specific information about UPF0458 protein C7orf42 homolog's disulfide bonds is not directly provided in the search results, related Xenopus proteins like XCGL-1 have been shown to form intermolecular disulfide bonds. In XCGL-1, cysteines at positions 18 and 35 are essential for the formation of disulfide-linked oligomers . By analogy, examination of the cysteine residues in UPF0458 protein might reveal similar important structural features.

A methodological approach would involve:

• Identifying all cysteine residues in the sequence

• Creating cysteine-to-alanine mutants

• Analyzing oligomeric states under reducing and non-reducing conditions by SDS-PAGE

• Performing mass spectrometry analysis to identify disulfide-linked peptides

Does the protein form oligomeric structures and what factors influence oligomerization?

Based on studies of related Xenopus proteins, it's reasonable to investigate whether UPF0458 protein forms oligomers. Factors that typically influence oligomerization include:

• Presence of specific cysteine residues: As seen with XCGL-1, specific cysteines (positions 18 and 35) are crucial for intermolecular disulfide bridge formation .

• Redox conditions: The ratio of oxidized to reduced glutathione during protein refolding can significantly affect oligomer formation.

• Calcium concentration: For proteins like XCGL-1, calcium is essential for proper folding and oligomerization .

• pH and ionic strength: These parameters can affect protein-protein interactions and should be optimized during refolding and storage.

How can recombinant UPF0458 protein be used in developmental biology studies?

Xenopus laevis is a powerful model for studying embryonic development. The recombinant UPF0458 protein can be utilized in:

• Gene expression studies: Analyzing the temporal and spatial expression patterns during development using antibodies against the recombinant protein.

• Loss-of-function studies: Designing morpholinos targeting UPF0458 mRNA and studying the resulting phenotypes. The availability of genome sequence data from Xenopus laevis helps in designing specific morpholinos while avoiding off-target effects .

• Gain-of-function studies: Introducing recombinant protein or mRNA into developing embryos to assess effects on developmental processes.

• Protein-protein interaction studies: Identifying binding partners during development using tagged recombinant protein for pull-down assays.

What experimental approaches are recommended for studying UPF0458 protein interactions with potential binding partners?

Several methodological approaches can be employed:

• Pull-down assays: Using tagged recombinant UPF0458 protein to isolate interaction partners from cell or tissue lysates.

• Yeast two-hybrid screening: For identifying direct protein-protein interactions.

• Bio-layer interferometry (BLI): This technique has been successfully used for studying binding kinetics of Xenopus proteins . It allows determination of association (ka) and dissociation (kd) rates, as well as binding affinity (KD).

• Cross-linking mass spectrometry: For identifying interaction interfaces within protein complexes.

• Co-immunoprecipitation: Using antibodies against UPF0458 to pull down protein complexes from Xenopus tissues or cells.

How does the 3D structure of UPF0458 protein influence its functional properties?

While specific structural data for UPF0458 protein C7orf42 homolog is not directly provided in the search results, molecular modeling approaches similar to those used for XCGL-1 can be applied . These include:

• Homology modeling: Using known structures of related proteins to predict the 3D structure.

• Electron microscopy: As demonstrated with XCGL-1, which adopted a four-lobed structure .

• Structure-function relationship studies: Creating targeted mutations in specific domains and assessing their impact on function.

• Molecular dynamics simulations: To predict protein flexibility and potential binding sites.

Understanding the structure would help elucidate how the protein's transmembrane domains are oriented and how they might participate in cellular functions.

What are the methodological considerations for analyzing differential expression of UPF0458 protein during Xenopus embryogenesis?

Analyzing expression patterns requires careful methodological planning:

• Stage-specific sampling: Collecting samples at defined developmental stages based on the standard Xenopus developmental table.

• RNA-seq analysis: Generating comprehensive expression profiles, as demonstrated in studies of Xenopus embryo regeneration . This approach can reveal stage-specific and tissue-specific expression patterns.

• Quantitative proteomics: Using techniques like SILAC or TMT labeling to quantify protein levels across developmental stages.

• In situ hybridization: For spatial localization of mRNA expression in intact embryos.

• Immunohistochemistry: Using antibodies against UPF0458 protein for protein localization studies.

For RNA-seq analysis, special consideration should be given to the wound-healing response in Xenopus embryos, which can complicate expression analysis. A stringent approach involving selection of genes increased by more than two-fold with respect to uncut embryos should be employed, as described in previous studies .

What are common challenges in maintaining protein stability during storage and experimentation?

Based on information from similar recombinant proteins, several considerations are important:

• Storage conditions: Store at -20°C/-80°C upon receipt. Aliquoting is necessary to avoid repeated freeze-thaw cycles .

• Buffer composition: Tris/PBS-based buffer with 50% glycerol at pH 8.0 has been shown to maintain stability .

• Reconstitution: It's recommended to reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added as a cryoprotectant .

• Working aliquots: For short-term use, store working aliquots at 4°C for up to one week .

How can researchers validate the proper folding and activity of recombinant UPF0458 protein?

Validation of proper folding and activity is crucial:

• Circular dichroism (CD) spectroscopy: To assess secondary structure elements.

• Thermal shift assays: To evaluate protein stability and proper folding.

• Functional assays: Developing specific activity assays based on predicted functions or by analogy with related proteins.

• Binding assays: If ligands are identified, bio-layer interferometry or surface plasmon resonance can validate binding functionality .

• Limited proteolysis: Properly folded proteins often show resistance to proteolytic digestion compared to misfolded variants.

How does Xenopus laevis UPF0458 protein differ from its homologs in other species?

Comparative analysis between species would involve:

• Sequence alignment: Comparing amino acid sequences of UPF0458 homologs from various species including rat, bovine, human, and zebrafish to identify conserved and divergent regions .

• Domain analysis: Identifying functional domains that may be conserved or species-specific.

• Phylogenetic analysis: Constructing evolutionary trees to understand the relationship between homologs across species.

• Expression pattern comparison: Analyzing whether expression patterns of homologs are conserved across species, suggesting conserved functions.

What can the Xenopus model system tell us about UPF0458 protein function that may be applicable to human research?

Xenopus offers several advantages as described in the literature :

• Developmental model: The external development of Xenopus embryos allows easy observation of developmental processes potentially influenced by UPF0458 protein.

• Genetic manipulation: Techniques like morpholino knockdown and CRISPR/Cas9 editing enable functional studies.

• Recombinant protein expression: The ability to express and characterize Xenopus UPF0458 protein can provide insights into the function of human homologs.

• Protein structure-function relationships: Insights gained from Xenopus protein studies may inform understanding of human homolog functions, particularly if structural elements are conserved.

• Disease modeling: As noted in the search results, "Findings from Xenopus research are highly applicable to human health and disease modeling as key biological processes are conserved" .

Reference Table: Properties of Recombinant Xenopus laevis UPF0458 protein C7orf42 homolog

Optimal Refolding Conditions for E. coli-Expressed UPF0458 protein

Data derived from refolding protocols for Xenopus proteins expressed in E. coli .