Recombinant Xenopus tropicalis BRO1 domain-containing protein BROX (brox)

What is the molecular structure of Xenopus tropicalis BROX protein?

Xenopus tropicalis BROX (BRO1 Domain and CAAX Motif Containing) is a 408 amino acid protein that contains a characteristic BRO1 domain. The full-length recombinant protein (AA 1-408) has been successfully expressed with a His tag in yeast expression systems. The complete amino acid sequence begins with MTHWFHRNPL and continues through a series of domains that maintain its structural and functional properties. The protein's sequence has been thoroughly characterized, providing the foundation for structure-function analyses in experimental settings .

Why is Xenopus tropicalis preferred over Xenopus laevis for BROX protein studies?

Xenopus tropicalis offers significant advantages for genetic and molecular studies of proteins like BROX due to its compact diploid genome (approximately 1.9 Gbp), in contrast to the pseudotetraploid genome of X. laevis. This genomic simplicity makes X. tropicalis particularly suitable for genetic manipulation and molecular characterization. Additionally, X. tropicalis has a shorter sexual maturation period (4-6 months) compared to X. laevis, enabling more rapid genetic experiments and transgenerational studies . When investigating proteins like BROX, the diploid nature of X. tropicalis simplifies genetic analyses, particularly when using CRISPR/Cas9 genome editing techniques, which have shown over 90% efficiency in targeting genes in somatic cells of founder animals .

How conserved is BROX protein between Xenopus tropicalis and humans?

While the search results don't explicitly state the conservation level of BROX specifically, Xenopus tropicalis as a model organism shares remarkable genetic similarity with humans. More than 79% of genes involved in human diseases have orthologous counterparts in X. tropicalis . This high level of conservation makes X. tropicalis BROX studies particularly relevant for understanding potential human BROX function. Comparative genomic analyses between species can reveal evolutionarily conserved domains and regulatory mechanisms that govern BROX function across vertebrates, providing insights into fundamental cellular processes .

How can I design effective CRISPR/Cas9 experiments to study BROX function in Xenopus tropicalis?

To effectively study BROX function using CRISPR/Cas9 in Xenopus tropicalis, researchers should first design specific guide RNAs targeting conserved functional domains of the BROX gene. The CRISPR components (guide RNAs and Cas9 mRNA or protein) can be microinjected into early-stage embryos, typically at the one-cell stage. For X. tropicalis specifically, the CRISPR-Cas9 method has demonstrated remarkable efficiency, disrupting target genes in more than 90% of somatic cells in founder animals . To verify editing efficiency, researchers should sequence the targeted region from genomic DNA extracted from embryo samples. For functional studies, both F0 "crispant" embryos and established knockout lines can be used depending on the experimental requirements. F0 analysis allows rapid screening while stable lines permit transgenerational and more detailed phenotypic studies .

What methods are recommended for purifying recombinant Xenopus tropicalis BROX protein?

For His-tagged recombinant Xenopus tropicalis BROX protein expressed in yeast systems, affinity chromatography using nickel or cobalt resins is the preferred initial purification method. This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE and Coomassie blue staining . For applications requiring higher purity, researchers should consider implementing additional purification steps such as size exclusion chromatography or ion exchange chromatography. When designing purification protocols, it's important to consider the protein's biochemical properties, including its theoretical isoelectric point and molecular weight. Additionally, optimization of buffer conditions (pH, salt concentration, and potential additives) is crucial for maintaining protein stability and activity during the purification process .

How can transgenic approaches be used to study BROX expression patterns in Xenopus?

Transgenic approaches provide powerful tools for studying BROX expression patterns in Xenopus. Researchers can employ BAC (Bacterial Artificial Chromosome) recombineering techniques to replace part of the BROX coding sequence with reporter genes like GFP. This approach allows visualization of BROX expression patterns in developing embryos. The I-SceI meganuclease method works efficiently for transgenesis in Xenopus tropicalis, enabling integration of transgenes into host chromosomes of founder animals with successful transmission to offspring . As demonstrated with other genes like Arx, recombineered BACs can faithfully recapitulate endogenous gene expression patterns when the BAC contains all necessary transcriptional regulatory elements . This approach is particularly valuable for studying dynamic expression patterns of BROX during development and in specific tissues.

What is known about the potential role of BROX in Xenopus tropicalis neural development?

While the search results don't specifically address BROX's role in neural development, Xenopus tropicalis serves as an excellent model for studying neurogenesis and brain development. Given that Xenopus models have been instrumental in discovering mechanisms of neural induction and patterning, investigating BROX expression and function in this context could yield valuable insights. Researchers can employ techniques such as in situ hybridization to characterize BROX expression in neural tissues, morpholino knockdown or CRISPR knockout to assess loss-of-function phenotypes, and gain-of-function approaches through mRNA injection to evaluate potential roles in neural specification or differentiation . The extensive toolkit available for Xenopus, including lineage tracing and explant cultures, makes it particularly suitable for dissecting the cellular and molecular functions of BROX in neural development .

How can I design experiments to investigate BROX protein interactions with the ESCRT pathway in Xenopus?

To investigate BROX interactions with the ESCRT (Endosomal Sorting Complex Required for Transport) pathway in Xenopus, researchers should first verify the conservation of ESCRT components between Xenopus and mammals. Based on the presence of the BRO1 domain in BROX, which is characteristic of proteins involved in multivesicular body formation, co-immunoprecipitation experiments using tagged recombinant BROX can identify binding partners within the ESCRT machinery. Researchers can also perform proximity labeling experiments (BioID or APEX) by expressing fusion proteins in Xenopus embryos or cell lines. In vivo significance can be assessed through co-localization studies using fluorescently tagged proteins and high-resolution microscopy. Functional studies might include examining the effects of BROX depletion or overexpression on ESCRT-dependent processes such as cytokinesis or autophagy in developing embryos .

What approaches can be used to study post-translational modifications of BROX in Xenopus tropicalis?

To study post-translational modifications (PTMs) of BROX in Xenopus tropicalis, researchers should employ a multi-faceted approach combining in silico prediction tools with experimental validation. Mass spectrometry analysis of immunoprecipitated endogenous BROX or purified recombinant protein can identify specific modification sites. Researchers can generate site-specific antibodies against predicted PTM sites or use PTM-specific detection methods. For functional analyses, site-directed mutagenesis of potential modification sites (e.g., changing phosphorylation-targeted serine/threonine residues to alanine or phosphomimetic aspartate/glutamate) followed by phenotypic assessment in embryos can reveal the significance of specific modifications. The effects of signaling pathway modulators on BROX modification status can provide insights into regulatory mechanisms. Additionally, researchers can examine temporal changes in BROX modifications throughout development or in response to specific stimuli .

How can comparative genomics approaches be used to understand BROX evolution and function across species?

Comparative genomics approaches offer powerful insights into BROX evolution and function across species. Researchers should begin by identifying BROX orthologs in diverse vertebrate and invertebrate species through reciprocal BLAST searches and synteny analysis. Multiple sequence alignment of these orthologs can reveal conserved domains and motifs that likely represent functionally important regions. Phylogenetic analysis can establish evolutionary relationships between BROX proteins and related BRO1 domain-containing proteins. Researchers can employ tools like PAML to detect signatures of positive selection, which might indicate adaptations in specific lineages. Comparison of gene expression patterns across species can identify conserved regulatory mechanisms. Finally, examination of protein interaction networks in different species can highlight evolutionarily conserved functional pathways involving BROX. These approaches collectively provide a comprehensive evolutionary context for understanding BROX function .

What are common challenges in expressing full-length Xenopus tropicalis BROX protein and how can they be addressed?

Common challenges in expressing full-length Xenopus tropicalis BROX protein include potential toxicity to host cells, protein misfolding, aggregation, and low yield. To address these issues, researchers should consider optimizing codon usage for the expression system, adjusting induction conditions (temperature, inducer concentration, duration), and testing different fusion tags beyond the standard His tag. For yeast expression systems, which have proven effective for BROX expression, selecting appropriate strain and media formulations is crucial. If protein toxicity is encountered, researchers might employ tightly controlled inducible promoters or expression of discrete functional domains rather than the full-length protein. For proteins with poor solubility, addition of solubility-enhancing tags (MBP, SUMO, etc.) or co-expression with chaperones may improve results. Finally, for applications requiring mammalian-like post-translational modifications, researchers should consider the trade-off between the economical yeast system and the more physiologically relevant but costly mammalian expression systems .

How can I validate the functional activity of purified recombinant Xenopus tropicalis BROX protein?

Validating the functional activity of purified recombinant Xenopus tropicalis BROX protein requires assays that assess both structural integrity and biological function. Initially, researchers should verify proper folding through circular dichroism spectroscopy or limited proteolysis. For BRO1 domain-containing proteins like BROX, binding assays with known interaction partners (particularly ESCRT pathway components) provide direct functional evidence. Researchers can develop in vitro assays measuring membrane binding or deformation activities, which are characteristic of BRO1 domain proteins. Additional validation approaches include testing the recombinant protein's ability to rescue phenotypes in BROX-depleted cells or embryos. If enzymatic activities are associated with BROX, specific activity assays should be developed. Finally, researchers should assess the protein's stability under various storage conditions to optimize preservation of functional activity for experimental applications .

What controls should be included when analyzing BROX expression patterns in Xenopus tropicalis embryos?

When analyzing BROX expression patterns in Xenopus tropicalis embryos, researchers should implement a comprehensive set of controls to ensure result validity. For in situ hybridization experiments, sense probe controls are essential to distinguish specific from non-specific staining. Researchers should include positive controls targeting genes with well-characterized expression patterns at the developmental stages being examined. When using antibodies to detect BROX protein, specificity should be validated through preabsorption tests and by demonstrating absence of signal in BROX-depleted embryos. For transgenic reporter approaches, multiple founder lines should be analyzed to control for position effects. Quantitative analyses of expression (qPCR, western blotting) should include reference genes or proteins shown to be stably expressed across the developmental stages being studied. Finally, correlating results from multiple detection methods (in situ hybridization, immunohistochemistry, reporter expression) provides the strongest evidence for genuine expression patterns .

How does BROX expression change during Xenopus tropicalis embryonic development?

While the search results don't provide specific information about BROX developmental expression patterns, researchers can employ multiple approaches to characterize these changes. Quantitative PCR using stage-specific RNA samples can establish the temporal profile of BROX expression, while whole-mount in situ hybridization can reveal spatial expression patterns. For higher resolution analysis, researchers can combine fluorescent in situ hybridization with confocal microscopy. Using the transgenic approaches successfully applied to other developmental genes in Xenopus, BROX regulatory elements can be linked to reporter genes to visualize expression dynamics in living embryos. Given that many developmental processes are conserved between Xenopus and other vertebrates, identifying when and where BROX is expressed during embryogenesis could provide important clues about its developmental functions. Researchers should examine expression at key developmental milestones including gastrulation, neurulation, and organogenesis, with particular attention to whether expression becomes restricted to specific tissues or cell types .

What can comparative analysis between Xenopus tropicalis and other vertebrate models tell us about conserved functions of BROX?

Comparative analysis between Xenopus tropicalis and other vertebrate models can illuminate evolutionarily conserved functions of BROX. Given that more than 79% of human disease genes have orthologs in X. tropicalis, there's likely significant functional conservation of BROX across vertebrates . Researchers should perform detailed sequence comparisons focusing on domain architecture preservation, conserved motifs, and key residues. Cross-species phenotypic comparisons following BROX disruption can reveal conserved developmental or cellular roles. Expression pattern comparisons across vertebrate models might identify conserved spatial-temporal regulation suggesting functional importance. The ability to perform efficient genome editing in multiple models, including Xenopus, zebrafish, and mice, enables direct functional comparisons. Additionally, rescue experiments, where BROX from one species is expressed in another species following endogenous BROX depletion, can definitively demonstrate functional conservation. These multi-model approaches provide robust evidence for core BROX functions maintained across vertebrate evolution .

What bioinformatic approaches are useful for analyzing BROX protein domains and predicting function?

Multiple bioinformatic approaches can be employed to analyze BROX protein domains and predict functions. Structure prediction tools (AlphaFold, I-TASSER) can generate three-dimensional models of BROX, particularly focusing on the BRO1 domain architecture. Researchers should use motif detection algorithms to identify functional elements beyond the major domains. Molecular dynamics simulations can predict protein flexibility and potential binding sites. For functional prediction, researchers should employ tools that identify structurally similar proteins (DALI server), predict protein-protein interactions (STRING database), and analyze potential post-translational modification sites. Gene Ontology enrichment analysis of predicted interaction partners can suggest biological processes involving BROX. Integration with transcriptomic data from Xenopus can identify co-expressed genes, potentially revealing functional networks. Finally, researchers should utilize databases of protein localization signals to predict subcellular distribution. As with all computational predictions, experimental validation remains essential for confirming BROX functions .

How can I integrate transcriptomic and proteomic data to understand BROX function in Xenopus tropicalis?

Integrating transcriptomic and proteomic data provides a comprehensive approach to understanding BROX function in Xenopus tropicalis. Researchers should begin by generating RNA-seq data from BROX-depleted or overexpressing embryos compared to controls, identifying differentially expressed genes that represent potential downstream targets or compensatory responses. In parallel, proteomic analysis using techniques like mass spectrometry can identify proteins whose levels change following BROX manipulation, as well as direct BROX interaction partners through co-immunoprecipitation or proximity labeling approaches. Integration of these datasets can be accomplished through pathway analysis tools that identify biological processes affected at both RNA and protein levels. Network analysis can reveal central nodes connecting BROX to its broader functional context. Researchers should pay particular attention to discordant RNA-protein relationships, which might indicate post-transcriptional regulation. Temporal analysis across developmental stages can further reveal dynamic aspects of BROX function. Finally, integration with existing developmental transcriptome atlases for Xenopus can place BROX-dependent processes in their proper developmental context .

What emerging technologies could advance our understanding of BROX function in Xenopus tropicalis?

Several emerging technologies hold promise for advancing our understanding of BROX function in Xenopus tropicalis. Single-cell RNA sequencing applied to developing embryos following BROX manipulation can resolve cell-type specific responses, potentially revealing functions in rare populations or transitional states. CRISPR activation/interference (CRISPRa/CRISPRi) systems, which modulate gene expression without altering the coding sequence, could allow temporal control of BROX expression. Base editing and prime editing technologies enable precise modification of specific residues to assess their functional importance. Optogenetic tools adapted for Xenopus could permit spatiotemporal control of BROX activity. Advanced imaging techniques, including light sheet microscopy and super-resolution approaches, can visualize BROX localization and dynamics with unprecedented detail. Proteomics advances, particularly proximity labeling methods optimized for developing embryos, can identify transient or compartment-specific BROX interactions. Finally, the integration of machine learning approaches with phenotypic data could identify subtle developmental abnormalities and predict functional relationships in BROX regulatory networks .

How might understanding BROX in Xenopus tropicalis contribute to human disease research?

Understanding BROX in Xenopus tropicalis could significantly contribute to human disease research through multiple avenues. Given that more than 79% of human disease genes have orthologs in X. tropicalis, mechanisms discovered in this model are likely relevant to human biology . If BROX functions in fundamental cellular processes like membrane trafficking or cytokinesis, these insights could illuminate pathological mechanisms in human disorders. The efficient CRISPR/Cas9 system in Xenopus tropicalis allows rapid modeling of human disease-associated BROX variants to assess their functional impacts. The accessibility of all developmental stages in Xenopus permits evaluation of BROX functions throughout development, potentially identifying critical periods relevant to developmental disorders. High-throughput screening approaches using Xenopus embryos could identify compounds that modulate BROX activity or rescue BROX-related phenotypes, suggesting therapeutic strategies. Additionally, interaction networks established in Xenopus could guide investigations of BROX-associated pathways in human cells, tissues, or clinical samples. This translational approach leverages the experimental advantages of Xenopus while maintaining focus on human health applications .

How should experiments be designed to distinguish between maternal and zygotic functions of BROX in Xenopus development?

Designing experiments to distinguish between maternal and zygotic functions of BROX in Xenopus development requires carefully timed interventions and specific analytical approaches. To analyze maternal contribution, researchers should employ antisense oligonucleotide injection into oocytes prior to fertilization to deplete maternal transcripts, or generate maternal-null females through CRISPR editing of primordial germ cells followed by host transfer. Temporal expression analysis using RT-qPCR and in situ hybridization can determine when the maternal-to-zygotic transition occurs for BROX. To specifically disrupt zygotic expression while preserving maternal contribution, researchers can use inducible CRISPR systems activated after the maternal-to-zygotic transition, or employ promoter-specific interference approaches. Rescue experiments are particularly informative: restoring BROX expression at different timepoints following depletion can reveal when BROX function is critically required. Additionally, researchers can perform embryo explant experiments where tissue is isolated before zygotic genome activation to assess purely maternal effects. These complementary approaches collectively provide a comprehensive understanding of stage-specific BROX functions .

How can researchers coordinate efforts to develop standardized resources for studying BROX in Xenopus tropicalis?

Researchers can coordinate efforts to develop standardized resources for studying BROX in Xenopus tropicalis through several collaborative approaches. Establishing a centralized repository for BROX reagents—including validated antibodies, expression constructs, and CRISPR guide RNAs—would ensure experimental reproducibility across laboratories. Development of reporter lines with fluorescently tagged endogenous BROX would provide valuable tools for localization and live imaging studies. Collaborative characterization of BROX knockout lines, with phenotypic data deposited in shared databases, would accelerate functional understanding. Researchers should establish standardized protocols for BROX protein expression, purification, and functional assays to enable direct comparison of results between groups. Integration with existing Xenopus resource centers, such as the National BioResource Project (NBRP) at Hiroshima University Amphibian Research Center, could provide infrastructure for distributing BROX-related resources . Regular workshops focused on technical advances and unpublished findings would foster open exchange of information. Finally, a dedicated online platform for sharing protocols, reagent validation data, and preliminary results would accelerate progress in the field .