Recombinant Xenopus tropicalis Vezatin (vezt)

What is Xenopus tropicalis Vezatin (vezt) and why is it important for research?

Vezatin (vezt) is a full-length protein (791 amino acids) encoded by the vezt gene in Xenopus tropicalis (Western clawed frog, also known as Silurana tropicalis). The protein is identified by UniProt accession number Q28C41 . Vezatin is important for research because it allows scientists to study protein function in a diploid amphibian model that offers several advantages over other systems. Xenopus tropicalis has emerged as a powerful model organism for combining genetic and genomic analysis with robust embryological, molecular, and biochemical assays . Its genome shows strong synteny with those of amniotes, making it valuable for comparative studies of protein function across vertebrate evolution . Additionally, the study of Vezatin in X. tropicalis can provide insights into cellular processes that may be relevant to understanding human disease mechanisms.

How is recombinant X. tropicalis Vezatin typically produced for research applications?

Recombinant X. tropicalis Vezatin is typically produced through heterologous expression in E. coli expression systems. The complete coding sequence (corresponding to amino acids 1-791) is cloned into an expression vector with an N-terminal His-tag to facilitate purification . The methodology involves:

• Cloning the full-length vezt gene (1-791 amino acids) into an appropriate expression vector

• Transforming the construct into a compatible E. coli strain

• Inducing protein expression under optimized conditions

• Lysing the bacterial cells and purifying the recombinant protein using affinity chromatography (typically with Ni-NTA resin that binds the His-tag)

• Performing additional purification steps as needed

• Lyophilizing the purified protein for longer-term storage

The resulting recombinant protein has the following properties:

• Full-length protein covering amino acids 1-791

• N-terminal His-tag for purification

• Greater than 90% purity as determined by SDS-PAGE

What are the recommended storage and handling conditions for recombinant X. tropicalis Vezatin?

For optimal stability and activity of recombinant X. tropicalis Vezatin, the following storage and handling recommendations should be followed:

Storage conditions:

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

• For longer-term storage, aliquoting is necessary to avoid repeated freeze-thaw cycles

• Working aliquots can 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% for long-term storage at -20°C to -80°C

Important handling notes:

• Repeated freezing and thawing is not recommended as it may lead to protein degradation

• The protein is typically shipped in a Tris/PBS-based buffer with 6% trehalose at pH 8.0

• For working solutions, a Tris-based buffer with 50% glycerol is often used to maintain stability

What are the advantages of using Xenopus tropicalis as a model system for studying proteins like Vezatin?

Xenopus tropicalis offers several significant advantages as a model system for studying proteins like Vezatin:

Genomic advantages:

• Diploid genome (unlike the tetraploid X. laevis), facilitating genetic analysis

• Relatively small genome size (~1.5×10⁹ bp), comparable to zebrafish

• Strong synteny with amniote genomes, simplifying orthology assignment

• Complete genome sequence available, facilitating genomic and genetic studies

Experimental advantages:

• High fecundity: a single pair can produce over 4000 embryos in a day

• External development allows easy observation and manipulation of embryos

• Embryos develop rapidly, with most major organs formed within 5 days

• Compatibility with many molecular and cellular techniques developed for X. laevis

• Amenable to genetic manipulation techniques, including CRISPR/Cas9 mutagenesis

• Shorter generation time compared to X. laevis (4-6 months vs. 1-2 years)

• Cost-effective maintenance compared to rodent models

Technical advantages:

• Many X. laevis tools can be applied without modification, including in situ hybridization protocols

• X. laevis probes often work in X. tropicalis, reducing the need to clone orthologs

• Antibodies against X. laevis proteins frequently cross-react with X. tropicalis proteins

• Amenable to antisense morpholino oligonucleotides for gene knockdown studies

• Unilateral mutagenesis at the 2-cell stage creates within-animal controls

These features make Xenopus tropicalis an excellent model for both basic research on protein function and translational studies relevant to human health and disease .

What methodological approaches can be used to study the function of X. tropicalis Vezatin in developmental contexts?

Investigating X. tropicalis Vezatin function in developmental contexts requires a multi-faceted approach combining genetic, biochemical, and imaging techniques:

Gene expression analysis:

• Temporal expression profiling: Using qPCR to quantify vezt expression throughout developmental stages. This can be normalized using housekeeping genes such as ornithine decarboxylase 1 (odc1) or ribosomal protein L8 (rpl8) .

• Spatial expression mapping: Employing whole-mount in situ hybridization to visualize vezt expression patterns in intact embryos. X. laevis protocols can be directly applied to X. tropicalis without modification .

• Single-cell RNA sequencing: To identify cell types expressing vezt during development and potential co-expression patterns with interacting genes.

Loss-of-function studies:

• CRISPR/Cas9 genome editing: Using microinjection of Cas9 protein and sgRNAs targeting the vezt locus. For developmental studies, injecting at the 2-cell stage allows creation of mosaic embryos with unilateral mutagenesis, providing an internal control .

• Morpholino knockdown: Designing antisense morpholino oligonucleotides to block vezt translation or splicing. This approach has been validated in X. tropicalis .

• Dominant negative approaches: Expressing truncated versions of Vezatin to interfere with endogenous protein function.

Gain-of-function studies:

• mRNA overexpression: Synthesizing capped mRNA encoding full-length or modified Vezatin for microinjection into embryos.

• Tissue-specific expression: Using the detailed fate map of X. tropicalis to target specific blastomeres for targeted expression in particular tissues .

Protein interaction studies:

• Co-immunoprecipitation: Using the recombinant His-tagged Vezatin to identify binding partners during development.

• Proximity labeling: Fusing Vezatin to BioID or TurboID for in vivo identification of proximal proteins.

• High-resolution imaging: Employing immunohistochemistry with anti-Vezatin antibodies to determine subcellular localization during development.

Phenotypic analysis:

• Morphological assessment: Examining impacts on organogenesis, tissue architecture, and cell migration.

• Behavioral assays: Evaluating effects on sensorimotor functions in later-stage tadpoles.

• Molecular phenotyping: Using RNA-seq or proteomics to identify downstream effects of Vezatin perturbation.

These approaches can be integrated to build a comprehensive understanding of Vezatin's developmental functions, potentially revealing new insights into cell adhesion, migration, or signaling pathways during embryogenesis.

What are the technical challenges in expressing and purifying functional X. tropicalis Vezatin in E. coli systems?

Expressing and purifying functional X. tropicalis Vezatin in E. coli presents several technical challenges that researchers should address through methodological optimization:

Expression challenges:

• Codon usage bias: The codon usage in X. tropicalis differs from E. coli, potentially leading to translational pausing or premature termination. This can be addressed by:

  • Codon optimization of the vezt sequence for E. coli

  • Using E. coli strains supplemented with rare tRNAs (e.g., Rosetta or CodonPlus strains)

  • Reducing expression temperature to allow more time for proper folding

• Protein solubility: Vezatin is a large protein (791 amino acids) that may form inclusion bodies in E. coli. Strategies to improve solubility include:

  • Using solubility-enhancing fusion tags (e.g., MBP, SUMO, or TrxA) in addition to the His-tag

  • Optimizing induction conditions (lower IPTG concentration, lower temperature)

  • Co-expressing molecular chaperones like GroEL/GroES

• Protein toxicity: If Vezatin interferes with bacterial processes, it may be toxic to the host cells. This can be mitigated by:

  • Using tightly controlled inducible promoters

  • Expression in bacterial strains with reduced proteolytic activity

  • Using auto-induction media for gradual protein expression

Purification challenges:

• Maintaining protein integrity: The full-length protein may be susceptible to degradation during purification. Approach:

  • Include protease inhibitors throughout the purification process

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Perform purification at 4°C to minimize proteolytic degradation

• Purification of His-tagged protein: Non-specific binding to Ni-NTA resin can reduce purity. Strategies include:

  • Optimizing imidazole concentration in washing steps

  • Including additional purification steps (ion exchange, size exclusion)

  • Using cobalt-based resins for more specific His-tag binding

• Protein refolding: If Vezatin forms inclusion bodies, refolding may be necessary:

  • Solubilize inclusion bodies with chaotropic agents (urea, guanidinium)

  • Use stepwise dialysis for gradual removal of denaturants

  • Add molecular chaperones during refolding process

Quality control assessments:

• Purity assessment: SDS-PAGE analysis to ensure >90% purity

• Structural integrity: Circular dichroism spectroscopy to verify secondary structure

• Functional assays: Developing binding assays to confirm that the recombinant protein retains its functional properties

By systematically addressing these challenges through methodological optimization, researchers can improve the yield and quality of recombinant X. tropicalis Vezatin for downstream applications.

How can genetic approaches be optimized for studying vezatin function in Xenopus tropicalis?

Optimizing genetic approaches for studying vezatin function in X. tropicalis requires careful consideration of experimental design and implementation strategies:

CRISPR/Cas9-based genome editing:

• sgRNA design optimization:

  • Target conserved functional domains of vezatin

  • Use X. tropicalis-specific genome browsers through Xenbase to identify optimal target sites

  • Employ multiple prediction algorithms to maximize on-target efficiency and minimize off-target effects

  • Design multiple sgRNAs targeting different exons to increase likelihood of functional disruption

• Delivery methods optimization:

  • For F0 analysis: microinjection of Cas9 protein with sgRNA into 1-cell or 2-cell stage embryos

  • For tissue-specific mutagenesis: target specific blastomeres based on the fate map

  • For germline transmission: inject dorsal animal blastomeres at 4-8 cell stage

• Mutation detection strategies:

  • T7 endonuclease I assay for initial screening

  • High-resolution melting analysis for rapid genotyping

  • Next-generation sequencing for precise characterization of mutations

  • PCR amplification and sequencing for confirming mutations at the vezatin locus, as demonstrated with other X. tropicalis genes

Conditional approaches:

• Inducible gene knockdown:

  • Design hormone-inducible constructs for temporal control

  • Use tissue-specific promoters for spatial regulation

  • Employ Cre-loxP or similar systems for conditional gene deletion

• Rescue experiments:

  • Co-inject wild-type vezatin mRNA with CRISPR components to validate specificity

  • Design rescue constructs resistant to sgRNA targeting for complementation studies

  • Create domain-specific deletions to map functional regions

Transgenic approaches:

• Reporter line generation:

  • Create vezatin-GFP fusion constructs to monitor subcellular localization

  • Develop vezt promoter-driven fluorescent reporters to track expression

  • Establish stable transgenic lines using transposon-based integration systems

• Enhancer/promoter analysis:

  • Identify and characterize vezatin regulatory elements

  • Use chromosome conformation capture techniques to identify long-range interactions

Genomic resource utilization:

• Leveraging X. tropicalis genomic resources:

  • Use the available genome assembly and annotation through Xenbase

  • Apply whole-exome enrichment technology for mutation identification

  • Utilize existing transcription factor catalogs to identify potential regulators of vezatin

• Comparative genomic analysis:

  • Examine conservation of vezatin across species to identify functional domains

  • Use synteny information to understand genomic context and potential co-regulated genes

By integrating these optimized genetic approaches, researchers can develop a comprehensive understanding of vezatin function in X. tropicalis, potentially revealing insights applicable to vertebrate biology more broadly.

What methods can be used to investigate the role of Vezatin in chromosome architecture and gene regulation in X. tropicalis?

Investigating Vezatin's potential role in chromosome architecture and gene regulation in X. tropicalis requires specialized methodologies that integrate genomic, molecular, and imaging approaches:

Chromosome conformation capture techniques:

• Hi-C analysis: To map genome-wide chromatin interactions and identify potential roles of Vezatin in maintaining or establishing topologically associating domains (TADs) .

  • Compare Hi-C maps between wild-type and vezatin-depleted embryos

  • Analyze changes in TAD boundaries and A/B compartmentalization

  • Identify specific genomic regions affected by Vezatin perturbation

• Chromosome conformation capture carbon copy (5C): For higher-resolution analysis of specific genomic regions.

• ChIP-seq integration: Combine Hi-C data with ChIP-seq of architectural proteins like CTCF and Rad21 to determine if Vezatin affects their binding or function .

Nuclear architecture analysis:

• Immunofluorescence microscopy:

  • Co-localization of Vezatin with nuclear landmarks

  • Analysis of nuclear morphology in vezatin mutants

  • Super-resolution imaging to determine precise subnuclear localization

• Electron microscopy:

  • Immunogold labeling to visualize Vezatin at ultrastructural level

  • Analysis of chromatin compaction states

• Live imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics

  • Single-molecule tracking of Vezatin-fluorescent protein fusions

Functional genomic approaches:

• RNA-seq after Vezatin perturbation:

  • Identify genes whose expression changes upon vezatin knockdown/knockout

  • Analyze whether affected genes cluster in specific genomic regions

  • Compare expression changes with alterations in chromatin architecture

• ATAC-seq or DNase-seq:

  • Map changes in chromatin accessibility

  • Correlate with gene expression changes and 3D genome organization

• CUT&RUN or CUT&Tag:

  • For high-resolution mapping of Vezatin binding sites

  • Identify co-factors binding near Vezatin sites

Developmental context integration:

• Stage-specific analysis:

  • Examine Vezatin's role during zygotic genome activation, which occurs before TAD establishment in X. tropicalis

  • Track changes in chromosome architecture throughout development

  • Compare with other model systems to identify conserved mechanisms

• Tissue-specific studies:

  • Analyze vezatin expression and function in different tissue contexts

  • Determine if Vezatin regulates tissue-specific gene expression programs

  • Compare TAD structures across different tissues

Chromatin remodeling connections:

• Interaction with chromatin remodelers:

  • Investigate potential physical interactions between Vezatin and the ISWI chromatin remodeling factor, which is required for de novo TAD formation

  • Examine functional connections through genetic interaction studies

By systematically applying these methodologies, researchers can determine whether Vezatin contributes to the establishment, maintenance, or dynamics of chromosome architecture and how this impacts gene regulation during X. tropicalis development.

What are the best practices for detecting and visualizing endogenous Vezatin expression patterns in X. tropicalis embryos?

For comprehensive detection and visualization of endogenous Vezatin expression patterns in X. tropicalis embryos, researchers should employ multiple complementary approaches:

mRNA expression detection:

• Whole-mount in situ hybridization (WISH):

  • Design antisense RNA probes specific to X. tropicalis vezatin transcripts

  • X. laevis WISH protocols can be applied directly to X. tropicalis without modification

  • Use of X. laevis probes may work if there is high sequence conservation

  • For increased sensitivity, employ RNAscope or similar methods for single-molecule detection

  • Process samples at multiple developmental stages to track temporal expression changes

• Section in situ hybridization:

  • For higher resolution analysis of expression in internal tissues

  • Combine with immunohistochemistry for co-localization studies

• Quantitative RT-PCR:

  • Design primers specific to X. tropicalis vezatin

  • Normalize to established reference genes such as ornithine decarboxylase 1 (odc1) or ribosomal protein L8 (rpl8)

  • Use comparative threshold cycle (CT) method to determine relative gene abundance

  • Sample multiple developmental stages and tissue types

Protein detection:

• Whole-mount immunohistochemistry:

  • Use antibodies specific to X. tropicalis Vezatin

  • X. laevis antibodies may cross-react due to high conservation

  • Verify specificity using vezatin knockdown/knockout controls

  • Optimize fixation and permeabilization for Vezatin epitope preservation

  • Use tyramide signal amplification for low-abundance detection

• Western blotting:

  • Extract proteins from different developmental stages

  • Use recombinant X. tropicalis Vezatin as a positive control

  • Ensure proper molecular weight detection (~87 kDa for the full-length protein)

  • Quantify relative expression levels across development

• Immunofluorescence on sections:

  • For subcellular localization and co-localization studies

  • Combine with tissue-specific markers

  • Use confocal or super-resolution microscopy for detailed localization

Reporter systems:

• BAC transgenesis:

  • Generate transgenic lines with GFP inserted into vezatin locus

  • Maintain large regulatory regions for authentic expression

  • Allow real-time visualization in living embryos

• Promoter analysis:

  • Clone putative vezatin promoter regions upstream of reporter genes

  • Identify minimal promoter elements sufficient for tissue-specific expression

Validation strategies:

• Morpholino knockdown controls:

  • Verify antibody specificity through vezatin knockdown

  • Confirm probe specificity by showing reduced signal in knockdown embryos

• CRISPR/Cas9 knockout controls:

  • Generate F0 CRISPR knockouts for validation

  • Create tissue-specific knockouts by targeting specific blastomeres

• Positive controls:

  • Use tissues with known Vezatin expression from other species

  • Include developmental stages with predicted high expression

By implementing these best practices, researchers can generate reliable, high-resolution data on the spatiotemporal expression pattern of Vezatin in X. tropicalis embryos, providing a foundation for functional studies of this protein during development.

How can researchers use recombinant X. tropicalis Vezatin to study protein interactions and biochemical functions?

Recombinant X. tropicalis Vezatin provides a powerful tool for investigating protein interactions and biochemical functions through a variety of in vitro and cell-based approaches:

Protein interaction discovery:

• Affinity purification-mass spectrometry (AP-MS):

  • Use His-tagged recombinant Vezatin as bait to pull down interacting proteins from X. tropicalis embryo lysates

  • Perform reciprocal pulldowns with candidate interactors

  • Identify interaction networks through bioinformatic analysis of MS data

• Yeast two-hybrid screening:

  • Use full-length Vezatin or specific domains as bait

  • Screen against X. tropicalis embryonic cDNA libraries

  • Validate positive interactions through secondary assays

• Protein microarrays:

  • Probe arrays containing X. tropicalis proteins with labeled recombinant Vezatin

  • Identify novel binding partners across the proteome

Interaction characterization:

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI):

  • Immobilize His-tagged Vezatin on sensor chips

  • Determine binding kinetics and affinities with putative partners

  • Investigate effects of mutations on binding properties

• Isothermal titration calorimetry (ITC):

  • Measure thermodynamic parameters of Vezatin interactions

  • Determine stoichiometry of complex formation

• Co-immunoprecipitation validation:

  • Express Vezatin with candidate interactors in heterologous systems

  • Confirm interactions in cellular contexts

  • Map interaction domains through deletion constructs

Structural studies:

• X-ray crystallography or cryo-EM:

  • Determine three-dimensional structure of Vezatin alone or in complex with partners

  • Identify critical residues involved in interactions

  • Guide structure-based functional studies

• Limited proteolysis:

  • Identify stable domains and flexible regions

  • Optimize construct design for structural studies

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational changes upon binding to partners

  • Identify regions involved in protein-protein interactions

Functional biochemical assays:

• In vitro binding assays:

  • ELISA-based binding studies using the recombinant protein

  • Pull-down assays with GST-fusion proteins or other tagged partners

  • Fluorescence polarization with labeled peptides from interaction domains

• Activity assays:

  • Investigate potential enzymatic functions

  • Test effects on known interactors' activities

  • Examine role in multiprotein complexes

• Cell-based functional studies:

  • Introduce recombinant Vezatin into cultured cells

  • Examine effects on cellular processes (adhesion, migration, etc.)

  • Perform competition assays with mutant versions

Antibody development:

• Generate Vezatin-specific antibodies:

  • Use recombinant protein as immunogen

  • Validate specificity in X. tropicalis tissues

  • Deploy for IP, ChIP, or imaging studies

• Epitope mapping:

  • Identify antibody binding sites on Vezatin

  • Develop epitope-specific antibodies for domain-specific studies

By systematically applying these approaches, researchers can build a comprehensive understanding of Vezatin's interaction network and biochemical functions, potentially revealing its roles in developmental processes, cellular architecture, or signaling pathways in X. tropicalis and other vertebrates.

What strategies can be employed to investigate potential roles of Vezatin in X. tropicalis development under stress conditions?

Investigating Vezatin's potential roles under stress conditions requires specialized experimental approaches that integrate developmental biology with stress response mechanisms:

Stress induction protocols:

• Environmental stressors:

  • Temperature stress: X. tropicalis embryos tolerate a narrower temperature range than X. laevis

  • Oxidative stress: Controlled H₂O₂ exposure

  • Osmotic stress: Altered media tonicity

  • UV irradiation: Controlled dosage experiments

  • pH stress: Acidification protocols similar to those used for TRPV1 studies

• Cellular stressors:

  • ER stress induction with tunicamycin or thapsigargin

  • Protein folding stress using proteasome inhibitors

  • DNA damage using chemical agents or irradiation

  • Energy stress through metabolic inhibitors

• Developmental challenges:

  • Hypoxia during critical developmental windows

  • Nutrient restriction models

  • Exposure to teratogens or environmental toxins

Gene expression analysis:

• Stress-responsive transcription:

  • Monitor vezatin expression changes under different stressors using qPCR

  • Compare with known stress-responsive genes like those identified in other Xenopus studies

  • Examine whether stress enhances vezatin transcription, similar to patterns observed for other genes in Xenopus

• Tissue-specific responses:

  • Perform in situ hybridization after stress exposure

  • Identify tissues with stress-induced vezatin expression changes

  • Compare with expression patterns of stress-response genes

Functional studies:

• Loss-of-function under stress:

  • CRISPR/Cas9 knockout or morpholino knockdown of vezatin

  • Expose embryos to stressors and assess survival, development, and recovery

  • Compare phenotypes between normal and stressed conditions

  • Evaluate stress resistance in vezatin-depleted embryos

• Gain-of-function experiments:

  • Overexpress Vezatin via mRNA microinjection before stress exposure

  • Test whether Vezatin overexpression provides protection against specific stressors

  • Identify domains required for stress-protective functions

Molecular mechanism investigations:

• Protein interaction shifts:

  • Compare Vezatin interactome under normal versus stress conditions

  • Identify stress-specific binding partners

  • Investigate potential interactions with known stress response factors

• Post-translational modifications:

  • Examine stress-induced phosphorylation, ubiquitination, or other modifications

  • Map modification sites and their functional consequences

  • Identify kinases or other enzymes that modify Vezatin during stress

• Subcellular localization changes:

  • Track Vezatin localization before, during, and after stress

  • Determine if stress triggers nuclear translocation or membrane redistribution

Cross-species comparative analysis:

• Functional analogy investigation:

  • Investigate potential functional similarities between X. tropicalis Vezatin and freeze-response proteins like the wood frog (Rana sylvatica) FR47 protein

  • Compare stress response mechanisms across amphibian species

  • Identify conserved stress-responsive domains