Recombinant Xenopus tropicalis UPF0458 protein C7orf42 homolog

What is Xenopus tropicalis UPF0458 protein C7orf42 homolog and what is its relationship to TMEM248?

The UPF0458 protein C7orf42 homolog from Xenopus tropicalis (Western clawed frog) is also known as transmembrane protein 248 (TMEM248). It is a 315-amino acid protein that functions as a transmembrane protein. The recombinant version is often expressed with an N-terminal His-tag in prokaryotic systems such as E. coli to facilitate purification and experimental applications . The protein is encoded by the tmem248 gene and is orthologous to human TMEM248. While its precise function remains under investigation, its conservation across species suggests it plays important biological roles that may be relevant to human health and disease .

Why is Xenopus tropicalis preferred as a model organism for studying this protein?

Xenopus tropicalis offers several significant advantages as a research model for studying TMEM248 and other proteins:

• Diploid genome: Unlike the related Xenopus laevis which is tetraploid, X. tropicalis has a diploid genome with high conservation to humans, making genetic manipulations and interpretations more straightforward .

• High synteny with human genome: The X. tropicalis genome exhibits considerable synteny with the human genome, facilitating the identification of orthologous genes and enabling translational research .

• Rapid development: Embryos develop quickly, with organ systems forming within 4 days, allowing for efficient phenotypic analysis .

• Cost-effectiveness: Maintaining X. tropicalis colonies is significantly less expensive than maintaining rodent models, making it accessible for more research groups .

• High fecundity: A single pair can produce over 4,000 embryos in a day, enabling large-scale, statistically powerful experiments .

These advantages make Xenopus tropicalis an excellent model for studying TMEM248 protein function and its potential role in human disease.

How is recombinant Xenopus tropicalis TMEM248 typically expressed and purified?

The recombinant Xenopus tropicalis TMEM248 protein is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification. The general expression and purification protocol includes:

• Expression vector construction: The full-length coding sequence (1-315aa) is cloned into an expression vector with an N-terminal His-tag .

• Bacterial transformation and culture: The expression construct is transformed into E. coli, followed by culture scale-up under optimized conditions.

• Protein expression induction: Expression is typically induced using IPTG or similar inducers.

• Cell lysis and initial purification: Bacterial cells are lysed, and the recombinant protein is captured using immobilized metal affinity chromatography (IMAC) via the His-tag.

• Further purification: Additional chromatography steps may be employed to achieve higher purity.

• Quality control: The final product typically achieves greater than 90% purity as determined by SDS-PAGE analysis .

The purified protein is then typically lyophilized for long-term storage and reconstituted in an appropriate buffer for experimental use.

What are the optimal storage conditions for recombinant Xenopus tropicalis TMEM248?

To maintain the stability and functionality of recombinant Xenopus tropicalis TMEM248, the following storage recommendations should be followed:

• Long-term storage: Store the lyophilized protein at -20°C to -80°C upon receipt .

• Aliquoting: Aliquoting is necessary for multiple uses to avoid repeated freeze-thaw cycles which can compromise protein integrity .

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

• Glycerol addition: Add 5-50% glycerol (final concentration) to aliquots for long-term storage at -20°C/-80°C, with 50% being the typical recommended concentration .

• Working aliquots: Store working aliquots at 4°C for up to one week .

• Storage buffer: The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

Adherence to these storage conditions will help maintain protein stability and activity for experimental applications.

How can CRISPR/Cas9 be used to study TMEM248 function in Xenopus tropicalis?

CRISPR/Cas9 mutagenesis in Xenopus tropicalis offers powerful approaches for studying TMEM248 function:

• Unilateral mutations: A unique advantage of Xenopus is the ability to create unilateral mutants by injecting only one cell at the 2-cell stage. This creates embryos with one half carrying homozygous mutations while the other half serves as an internal control, enabling direct side-by-side comparison within the same animal .

• F0 generation analysis: Unlike many model organisms that require breeding to homozygosity, Xenopus tolerates high efficiency mutagenesis in F0 embryos, allowing rapid analysis without the need for germline transmission .

• Multiplexed gene analysis: The ease of generating thousands of mutant embryos makes it feasible to study multiple genes in parallel, which is particularly valuable for identifying shared phenotypes among disease-associated genes .

• Variant modeling: Beyond simple knockouts, CRISPR can be used with homology-directed repair to introduce specific missense variants of interest to model human disease mutations .

• Tissue-specific targeting: The well-characterized fate map of early Xenopus embryos allows targeting of genetic perturbations to specific tissues .

These approaches can efficiently elucidate TMEM248 function in development and disease contexts.

What phenotypic assays are most informative when studying TMEM248 function in Xenopus?

When investigating TMEM248 function in Xenopus tropicalis, several phenotypic assays provide valuable insights:

• Brightfield microscopy: Initial whole-animal examination to identify gross morphological changes, which is particularly important for relating phenotypes to expression patterns and human clinical presentations .

• In situ hybridization: To identify tissues and developmental stages where TMEM248 is expressed, guiding subsequent phenotypic analysis .

• Immunohistochemistry: For analyzing protein localization and potential disruptions in cellular architecture.

• Behavioral assays: Quantitative behavioral analyses by day 10 of development can serve as functional readouts for neurological phenotypes .

• Organogenesis assessment: Detailed examination of organ development, which can be accomplished through tissue-specific transgenic reporter lines .

• Transcriptomic analysis: Single-cell or bulk RNA sequencing to identify molecular consequences of TMEM248 perturbation .

• Electron microscopy: For ultrastructural analysis, particularly relevant for transmembrane proteins.

These complementary approaches can provide a comprehensive understanding of TMEM248 function across multiple levels of biological organization.

How does the Xenopus tropicalis TMEM248 compare to human TMEM248 in terms of conservation?

Understanding the evolutionary conservation between Xenopus tropicalis TMEM248 and human TMEM248 is crucial for translational research. While the search results don't provide direct sequence comparison data, several factors support the value of using Xenopus tropicalis as a model:

• Genome conservation: The Xenopus tropicalis genome shows high conservation with the human genome, with well-preserved synteny making orthologous gene identification straightforward .

• Functional domains: Transmembrane domains and functional motifs are typically highly conserved across vertebrates, suggesting that insights from Xenopus studies may be applicable to human biology.

• Diploid advantage: The diploid nature of Xenopus tropicalis (versus the tetraploid Xenopus laevis) makes it more suitable for studying gene function with direct relevance to humans .

• Disease modeling capacity: The proven track record of Xenopus tropicalis in modeling human genetic disorders suggests that TMEM248 function is likely to be sufficiently conserved for translational insights .

Researchers should perform detailed sequence alignment and structural prediction analyses when designing experiments to account for any species-specific differences.

What approaches can be used to investigate protein-protein interactions of TMEM248?

To elucidate the interactome of Xenopus tropicalis TMEM248, researchers can employ several complementary approaches:

• Co-immunoprecipitation: Using the His-tagged recombinant protein as bait to identify binding partners in Xenopus tissue lysates.

• Proximity labeling: BioID or APEX2 fusions to TMEM248 expressed in Xenopus embryos can identify proximal proteins in living cells.

• Yeast two-hybrid screening: Using the cytoplasmic domains of TMEM248 as bait to identify potential interacting proteins.

• Mass spectrometry-based interactomics: Quantitative proteomics comparing wild-type and TMEM248-deficient samples to identify altered protein complexes.

• Xenopus ORFeome resources: Leveraging the comprehensive set of Xenopus ORF clones available through the NICHD-funded Xenopus ORFeome project for high-throughput interaction screening .

• Fluorescence resonance energy transfer (FRET): To validate direct protein-protein interactions in living cells.

• Surface plasmon resonance: Using purified recombinant proteins to quantify binding kinetics and affinities.

These approaches can provide insights into the molecular function of TMEM248 through its interaction network.

How can variant effects be assessed using Xenopus tropicalis TMEM248?

Xenopus tropicalis provides an excellent platform for modeling and assessing the effects of TMEM248 variants:

• Variant introduction: CRISPR/Cas9-mediated homology-directed repair can be used to introduce specific variants of interest at the endogenous locus .

• Rescue experiments: Wild-type and variant mRNAs can be co-injected with CRISPR/Cas9 components targeting the endogenous gene to assess rescue capabilities.

• Variant classification: Comparing phenotypes between known pathogenic variants and variants of uncertain significance (VUS) can aid in clinical interpretation .

• Cross-species validation: Phenotypic consistency between human patients and Xenopus models provides strong evidence for variant pathogenicity .

• Mechanistic insight: Detailed phenotypic and molecular analysis can reveal the specific biological processes disrupted by particular variants.

• Therapeutic testing: Once variant effects are understood, Xenopus models can be used to screen potential therapeutic approaches.

This approach is particularly valuable for variants in highly conserved regions of TMEM248 where Xenopus and human proteins share significant structural and functional similarity.

What technical considerations are important when using recombinant TMEM248 for in vitro studies?

When utilizing recombinant Xenopus tropicalis TMEM248 for in vitro experiments, several technical considerations are critical:

• Reconstitution protocol: Centrifuge the vial briefly before opening to bring contents to the bottom. Reconstitute in deionized sterile water to 0.1-1.0 mg/mL .

• Glycerol addition: Add 5-50% glycerol to stabilize the protein for storage, with 50% being the default recommendation .

• Freeze-thaw avoidance: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided .

• Buffer compatibility: The protein is provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which may need to be considered for downstream applications .

• Purity assessment: The recombinant protein typically has >90% purity as determined by SDS-PAGE, but specific experiments may require additional purification steps .

• Detergent considerations: As a transmembrane protein, TMEM248 may require appropriate detergents for solubility in certain applications.

• Post-translational modifications: E. coli-expressed proteins lack eukaryotic post-translational modifications, which should be considered when interpreting functional data.

Attention to these technical details will optimize experimental outcomes when working with recombinant TMEM248.