Recombinant Xenopus tropicalis Eukaryotic peptide chain release factor subunit 1 (etf1)

What is Xenopus tropicalis etf1 and why is it significant for research?

Xenopus tropicalis etf1 (also known as erf1 or cl1) is the eukaryotic translation termination factor 1 that functions as a peptide chain release factor in protein synthesis. It directs the termination of nascent peptide synthesis in response to the termination codons UAA, UAG, and UGA. Additionally, it serves as a component of the transient SURF complex which recruits UPF1 to stalled ribosomes .

The significance of studying X. tropicalis etf1 lies in the advantages of this model organism for genetic and molecular research. Unlike its close relative X. laevis (which is tetraploid), X. tropicalis has a diploid genome with greater conservation to mammalian gene structure and function. This makes it particularly valuable for comparative gene function and regulation studies . The high level of genomic synteny between X. tropicalis and humans facilitates straightforward identification of orthologous genes, making it an excellent model for understanding fundamental mechanisms of translation termination and protein synthesis .

How does the structure of X. tropicalis etf1 compare to human etf1?

A typical comparison table for protein conservation might include:

What expression patterns does etf1 show during X. tropicalis development?

Based on the general information about X. tropicalis as a model organism, developmental gene expression can be studied using in situ hybridization techniques that allow visualization of temporal and spatial expression patterns . While specific etf1 expression data isn't provided in the search results, researchers commonly examine expression across developmental stages, embryonic tissues, and adult tissues as indicated by the Xenbase database structure .

Understanding developmental expression patterns is crucial for researchers investigating etf1's role in early development, tissue differentiation, and cellular functions. The well-established developmental fate map of early X. tropicalis embryos facilitates precise correlation between gene expression and developmental outcomes .

What techniques are most effective for cloning X. tropicalis etf1 for recombinant expression?

For effective cloning of X. tropicalis etf1, researchers should consider:

• Utilizing the accurate, annotated reference genome available through Xenbase (https://www.xenbase.org) to design appropriate primers for PCR amplification .

• Extracting high-quality RNA from appropriate X. tropicalis developmental stages or tissues where etf1 is known to be expressed, followed by RT-PCR to obtain the full-length cDNA.

• Employing gateway cloning or traditional restriction enzyme cloning methods to insert the etf1 cDNA into an appropriate expression vector. For bacterial expression, pET-series vectors are commonly used, while mammalian expression might utilize CMV promoter-driven vectors.

• Confirming sequence integrity through comprehensive sequencing to ensure no mutations were introduced during the cloning process.

• Optimizing codons if necessary, particularly when expressing in bacterial systems, as eukaryotic codon usage differs from prokaryotic systems.

What expression systems are optimal for producing recombinant X. tropicalis etf1 protein?

The choice of expression system depends on research requirements, with each system offering distinct advantages:

Bacterial Expression (E. coli):

• Advantages: High yield, cost-effective, rapid production

• Considerations: May lack post-translational modifications; protein folding issues may occur

• Recommendation: Optimal for structural studies or applications not requiring post-translational modifications

• Methodology: Use of T7 promoter-based vectors (pET series) with BL21(DE3) or Rosetta strains; expression at lower temperatures (16-20°C) may improve protein folding

Insect Cell Expression:

• Advantages: Eukaryotic post-translational modifications; better protein folding than bacterial systems

• Methodology: Baculovirus expression vector systems using Sf9 or High Five cells

• Applications: Functional studies requiring properly folded protein with some post-translational modifications

Mammalian Expression:

• Advantages: Most authentic post-translational modifications and protein folding

• Methodology: Transient transfection of HEK293 or CHO cells; stable cell line generation for consistent production

• Applications: Functional assays requiring native-like protein conformation

Xenopus Oocyte Expression:

• Advantages: Native context for the protein; useful for functional studies

• Methodology: Microinjection of mRNA into oocytes

• Applications: Electrophysiological studies or cellular localization analyses

What purification strategies yield the highest purity recombinant X. tropicalis etf1?

A comprehensive purification strategy for recombinant X. tropicalis etf1 typically involves:

• Affinity Chromatography:

  • His-tag purification using Ni-NTA resin is common for initial capture

  • Alternative tags include GST, FLAG, or MBP depending on downstream applications

  • Critical parameters: Imidazole concentration in wash buffers (typically 20-50 mM) and elution buffers (250-500 mM)

• Secondary Purification:

  • Ion exchange chromatography based on etf1's isoelectric point

  • Size exclusion chromatography to remove aggregates and ensure monodispersity

  • Typical yields: 5-15 mg/L in bacterial systems; 1-5 mg/L in insect cell systems

• Quality Control Assessments:

  • SDS-PAGE to verify size and initial purity (>90% is typically achievable)

  • Western blotting to confirm identity

  • Dynamic light scattering to assess monodispersity

  • Functional assays to confirm activity (translation termination assays)

• Storage Considerations:

  • Buffer optimization to maintain stability (typically phosphate or Tris buffers with 150-300 mM NaCl)

  • Addition of glycerol (10-20%) for freezing

  • Flash freezing in liquid nitrogen and storage at -80°C in small aliquots

How can CRISPR/Cas9 be used to study etf1 function in X. tropicalis?

CRISPR/Cas9 genome editing in X. tropicalis provides powerful approaches to study etf1 function:

• Knockout Generation:

  • Design sgRNAs targeting early exons of etf1

  • Microinject CRISPR/Cas9 components (sgRNA and Cas9 protein or mRNA) into fertilized eggs at the one-cell stage

  • X. tropicalis is particularly amenable to CRISPR/Cas9 editing, with high efficiency reported

  • Screening for mutations can be performed by T7 endonuclease assay, HRMA, or direct sequencing

• Unilateral Mutant Strategy:

  • A unique advantage of X. tropicalis is the ability to create unilateral mutants by injecting one cell at the two-cell stage

  • This creates embryos with one side carrying the mutation while the other side serves as an internal control

  • This approach is especially valuable for studying essential genes where complete knockout might be lethal

• Phenotypic Analysis:

  • Assess effects on embryonic development, particularly protein synthesis-dependent processes

  • Examine tissue-specific requirements using the unilateral approach

  • Temporal requirements can be studied using inducible CRISPR systems

• Precise Editing Applications:

  • Introduction of specific patient variants to model disease-associated mutations

  • Tagged endogenous protein generation for localization studies

  • Critical methodological consideration: HDR (homology-directed repair) template design with ~800-1000 bp homology arms

What functional assays can assess the activity of recombinant X. tropicalis etf1?

Several functional assays can be employed to assess etf1 activity:

• In vitro Translation Termination Assays:

  • Cell-free translation systems using rabbit reticulocyte lysate or wheat germ extract

  • Reporter constructs containing stop codons (UAA, UAG, UGA) followed by luciferase or other measurable reporters

  • Readthrough efficiency measurement with dual reporter systems

  • Quantitative metrics: Percent termination efficiency, kinetic parameters (kcat/KM)

• SURF Complex Formation Analysis:

  • Co-immunoprecipitation assays to detect interactions with other SURF complex components

  • Size exclusion chromatography to verify complex formation

  • Surface plasmon resonance to determine binding kinetics and affinities

• Cell-Based Assays:

  • Transfection of etf1-depleted cells with wild-type or mutant etf1 to assess rescue of translation defects

  • Reporter systems containing premature termination codons to assess nonsense-mediated mRNA decay activity

  • Polysome profiling to examine effects on global translation

• X. tropicalis Embryo Rescue Experiments:

  • Morpholino or CRISPR-mediated depletion of endogenous etf1 followed by rescue with recombinant protein or mRNA

  • Quantification of phenotypic rescue provides functional validation

  • Advantage: Assesses function in the native organism context

How can recombinant X. tropicalis etf1 be used to study nonsense-mediated decay mechanisms?

Recombinant X. tropicalis etf1 provides valuable tools for dissecting nonsense-mediated decay (NMD) mechanisms:

• Reconstitution of SURF Complex Formation:

  • Etf1 is a component of the SURF complex that recruits UPF1 to stalled ribosomes

  • In vitro reconstitution with purified components allows mechanistic studies of complex assembly

  • Mutational analysis of interaction domains can identify critical residues

  • Methodological approach: Pull-down assays using tagged etf1 combined with mass spectrometry to identify interacting partners

• Structure-Function Analysis:

  • Site-directed mutagenesis of conserved residues to determine their role in stop codon recognition and UPF1 recruitment

  • Truncation constructs to map functional domains

  • Combining with cryoEM analysis to determine structural conformations during termination and NMD initiation

• Comparing Wild-Type and Variant Activities:

  • Analysis of naturally occurring variants or disease-associated mutations

  • Measurement of termination efficiency versus readthrough propensity

  • Kinetic analysis of the termination reaction using pre-steady state kinetics

• X. tropicalis as a Model System:

  • The diploid nature of X. tropicalis makes it an excellent system for studying NMD mechanisms in vivo

  • Injection of reporter constructs containing premature termination codons (PTCs) with wild-type or mutant etf1

  • Quantification of mRNA decay rates and protein expression levels

What structural insights can be gained from studying X. tropicalis etf1 compared to other species?

Structural comparative analysis of X. tropicalis etf1 can provide valuable evolutionary and functional insights:

• Evolutionary Conservation Analysis:

  • Mapping conserved residues across species identifies functionally critical regions

  • X. tropicalis provides an excellent intermediate evolutionary position between mammals and more distant vertebrates

  • The diploid nature of X. tropicalis simplifies structural genomic analysis compared to tetraploid species like X. laevis

• Structure Determination Approaches:

  • X-ray crystallography of recombinant X. tropicalis etf1 alone or in complex with release factors or ribosomal components

  • CryoEM analysis of etf1-ribosome complexes at different stages of termination

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon binding partners

• Comparative Structure-Function Analysis:

  • Chimeric constructs between X. tropicalis and human etf1 to map species-specific functional domains

  • Structural basis for differences in stop codon recognition efficiency between species

  • Analysis of post-translational modification sites and their functional consequences

• Methodological Considerations:

  • Expression of isotopically labeled protein for NMR studies

  • Optimization of crystallization conditions using truncated constructs if full-length protein proves challenging

  • Computational modeling and molecular dynamics simulations to predict functional motions

How does etf1 function in the context of X. tropicalis developmental biology?

Understanding etf1's role in X. tropicalis development provides insights into fundamental translational control mechanisms:

• Temporal Expression Analysis:

  • Quantitative RT-PCR analysis across developmental stages

  • Western blot analysis to correlate mRNA with protein levels

  • Single-cell transcriptomic data analysis to identify cell-type specific expression patterns

  • X. tropicalis developmental transcriptomics datasets can be mined from Xenbase

• Spatial Expression Studies:

  • In situ hybridization to localize etf1 mRNA in developing embryos

  • Immunohistochemistry with anti-etf1 antibodies to detect protein localization

  • Integration with fate mapping data to correlate expression with developmental trajectories

• Loss-of-Function Approaches:

  • CRISPR/Cas9 knockout or knockdown studies using the unilateral injection method

  • Morpholino-mediated knockdown for stage-specific analyses

  • Assessment of developmental defects in protein synthesis-dependent processes

  • Rescue experiments with wild-type or mutant etf1 to validate specificity

• Translational Control Analysis:

  • Polysome profiling in etf1-depleted embryos to assess global translation effects

  • Ribosome profiling to identify specific mRNAs affected by etf1 perturbation

  • Integration with proteomics to correlate transcriptome and proteome changes

How can X. tropicalis etf1 be used to model human diseases associated with translation termination defects?

X. tropicalis provides an excellent platform for modeling human diseases related to translation termination:

• Disease Variant Modeling:

  • CRISPR/Cas9-mediated introduction of patient-specific variants into the endogenous X. tropicalis etf1 gene

  • Expression of disease-associated variants in etf1-depleted backgrounds

  • X. tropicalis has proven effective at recapitulating human disease phenotypes, sometimes more accurately than rodent models

• Readthrough Therapy Screening:

  • Screening compounds that promote readthrough of premature termination codons

  • Assessment of specificity using different stop codon contexts

  • Advantage of X. tropicalis: Large-scale drug screening is facilitated by embryos absorbing small molecules from culture medium

• Mechanistic Studies of Disease Pathways:

  • Analysis of downstream effects on specific transcripts containing disease-relevant termination codons

  • Investigation of tissue-specific requirements for accurate translation termination

  • Integration with transcriptomics and proteomics to identify global consequences

• Therapeutic Strategy Development:

  • Testing gene therapy approaches using X. tropicalis as a rapid screening platform

  • Assessment of antisense oligonucleotides targeting specific transcripts

  • Evaluation of small molecules that modulate etf1 activity or interactions

What challenges arise in expressing and purifying recombinant X. tropicalis etf1, and how can they be addressed?

Common challenges and their solutions include:

• Protein Solubility Issues:

  • Challenge: Recombinant etf1 may form inclusion bodies in bacterial systems

  • Solutions:

    • Expression at lower temperatures (16-20°C)

    • Use of solubility-enhancing tags (MBP, SUMO)

    • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

    • Optimization of induction conditions (IPTG concentration, OD at induction)

• Protein Stability Issues:

  • Challenge: Purified etf1 may show degradation or aggregation

  • Solutions:

    • Buffer optimization (screening different pH, salt concentrations, additives)

    • Addition of protease inhibitors throughout purification

    • Thermal shift assays to identify stabilizing buffer conditions

    • Size exclusion chromatography to remove aggregates

• Functional Activity Preservation:

  • Challenge: Loss of activity during purification

  • Solutions:

    • Gentle purification methods avoiding harsh conditions

    • Activity assays at each purification step to track activity

    • Addition of stabilizing co-factors or binding partners

    • Minimizing freeze-thaw cycles by aliquoting and flash-freezing

• Post-Translational Modifications:

  • Challenge: Bacterial systems lack eukaryotic PTM machinery

  • Solutions:

    • Use of eukaryotic expression systems for studies requiring PTMs

    • Mass spectrometry analysis to identify and characterize PTMs

    • In vitro enzymatic addition of specific modifications when possible

How can researchers resolve discrepancies in experimental results when working with recombinant X. tropicalis etf1?

When facing experimental discrepancies, systematic troubleshooting approaches include:

• Protein Quality Assessment:

  • Verify protein purity by SDS-PAGE and mass spectrometry

  • Confirm protein folding using circular dichroism or fluorescence spectroscopy

  • Assess aggregation state using dynamic light scattering or analytical ultracentrifugation

  • Check for truncations or degradation using western blotting with antibodies against different regions

• Experimental Variable Control:

  • Standardize protein concentrations using accurate quantification methods (BCA, Bradford)

  • Control buffer conditions precisely (pH, ionic strength, reducing agents)

  • Monitor and control temperature during experiments

  • Use positive and negative controls in all assays

• Assay Validation:

  • Verify assay sensitivity and dynamic range using known controls

  • Perform dose-response experiments to ensure linearity

  • Test multiple assay methods to cross-validate results

  • Consider interference from tags or other components

• Statistical Analysis:

  • Perform sufficient biological and technical replicates

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to determine adequate sample sizes

  • Report variability and confidence intervals

What are the limitations of using X. tropicalis etf1 as a model for human etf1 function?

While X. tropicalis provides many advantages, researchers should be aware of potential limitations:

• Evolutionary Divergence:

  • Despite high conservation, species-specific differences in etf1 function may exist

  • Some protein-protein interactions may differ between amphibian and human systems

  • Methodological solution: Comparative functional assays between X. tropicalis and human etf1 proteins

• Physiological Context Differences:

  • Temperature optima differ (X. tropicalis is adapted to lower temperatures)

  • Cell type-specific functions may not be fully conserved

  • Methodological approach: Validation of key findings in mammalian cell culture systems

• Developmental Timing Variations:

  • Developmental programs and timing differ between amphibians and mammals

  • Some developmental processes requiring etf1 may be species-specific

  • Solution: Careful interpretation and validation when extrapolating developmental findings

• Technical Considerations:

  • Antibody cross-reactivity issues between species

  • Different codon usage preferences may affect recombinant expression

  • Methodological approach: Generate species-specific tools and reagents when necessary

How might integrating multi-omics approaches enhance our understanding of X. tropicalis etf1 function?

Multi-omics integration offers powerful approaches to comprehensively understand etf1 function:

• Integrative Genomics and Proteomics:

  • Combination of ribosome profiling, RNA-seq, and proteomics in etf1-depleted embryos

  • Identification of transcripts most sensitive to etf1 perturbation

  • Correlation between changes in transcript abundance and protein levels

  • Analysis of ribosome occupancy at termination codons to identify affected genes

• Structural Biology Integration:

  • Combining cryoEM, X-ray crystallography, and computational modeling

  • Integration with cross-linking mass spectrometry to map interaction interfaces

  • Correlation of structural features with functional outcomes from mutagenesis

  • Development of structure-based predictive models for etf1 activity

• Single-Cell Approaches:

  • Single-cell transcriptomics to identify cell type-specific requirements for etf1

  • Spatial transcriptomics to map etf1 activity patterns in developing embryos

  • Integration with lineage tracing to connect etf1 function to cell fate decisions

  • X. tropicalis embryos are particularly well-suited for these approaches due to their well-characterized developmental map

• Network Analysis:

  • Construction of protein-protein interaction networks centered on etf1

  • Integration with genetic interaction data from genome-wide screens

  • Machine learning approaches to predict etf1-dependent processes

  • Systems biology modeling of translation termination dynamics

What novel technologies might advance research on X. tropicalis etf1 function?

Emerging technologies with potential to advance etf1 research include:

• CRISPR-Based Technologies:

  • Base editing for precise introduction of point mutations without double-strand breaks

  • CRISPRi/CRISPRa for reversible modulation of etf1 expression

  • CRISPR screening approaches to identify genetic interactors

  • X. tropicalis has demonstrated high efficiency with CRISPR technologies

• Imaging Advances:

  • Live imaging of translation termination events using fluorescent reporter systems

  • Super-resolution microscopy to visualize etf1 localization and dynamics

  • Optogenetic approaches to control etf1 activity with spatiotemporal precision

  • Correlative light and electron microscopy to connect molecular events with ultrastructural context

• Protein Engineering:

  • Split protein complementation assays to monitor etf1 interactions in vivo

  • Development of biosensors to detect translation termination efficiency

  • Engineered etf1 variants with enhanced or altered specificities

  • Nanobodies or aptamers to modulate etf1 function with high specificity

• In vitro Reconstitution:

  • Cell-free expression systems to study translation termination in controlled environments

  • Microfluidic approaches for high-throughput analysis of termination kinetics

  • Reconstituted minimal translation systems to dissect mechanistic details

  • Single-molecule approaches to observe individual termination events