Recombinant Xenopus laevis Protein FAM73A (fam73a)

What is FAM73A protein and what is its significance in Xenopus laevis?

FAM73A (Family with sequence similarity 73 member A) is a protein encoded by the fam73a gene in Xenopus laevis (African clawed frog). The full-length protein consists of 570 amino acids and contains specific domains involved in cellular functions. The significance of studying this protein in Xenopus laevis stems from the frog's position as an important model organism for developmental and cell biology research. Xenopus egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms that are conserved in humans, making this system valuable for comparative studies of protein function .

How does the amino acid sequence of Xenopus laevis FAM73A compare to mammalian homologs?

The Xenopus laevis FAM73A protein (UniProt: Q6NRB7) has a sequence of 570 amino acids that begins with "MTETQHIFRLTVHRFMDFPLSIYSSFTQLKPTPGLKKIIAVAAISGVSLIFLACHLKRKR..." as detailed in the database . Comparative analysis with mammalian homologs shows conserved domains, particularly in regions associated with membrane localization and protein-protein interactions. The protein contains transmembrane domains suggested by the sequence "IIAVAAISGVSLIFLACHLKRKR," indicating it likely functions as a membrane protein . This conservation across species suggests evolutionary importance of its function.

What are the predicted structural domains of Xenopus laevis FAM73A protein?

Based on sequence analysis, Xenopus laevis FAM73A contains several predicted structural domains:

• N-terminal transmembrane domain (amino acids approximately 28-50)

• Multiple coiled-coil domains throughout the protein

• Several phosphorylation sites predicted in the C-terminal region

• Conserved "IFLMGTGRK" motif (amino acids 340-348) that may be involved in nucleotide binding

The protein's structural architecture suggests roles in membrane organization and potential involvement in cellular signaling pathways.

How is FAM73A expressed during Xenopus development?

FAM73A expression in Xenopus follows a specific developmental pattern. While comprehensive expression data is not fully detailed in the available sources, studies using deep proteomics approaches have identified FAM73A in unfertilized Xenopus eggs, suggesting maternal inheritance of this protein . The protein likely plays roles in early development before zygotic gene expression begins. Expression patterns can be studied using approaches similar to those used in the proteomics analysis of Xenopus eggs, which have identified more than 11,000 proteins with 99% confidence .

What experimental systems are best suited for studying Xenopus laevis FAM73A?

Several experimental systems are ideal for studying Xenopus laevis FAM73A:

• Xenopus egg extracts: These cell-free systems contain all factors required for complex biological processes and are particularly useful for studying protein function outside of cells .

• Recombinant protein expression systems: E. coli expression systems can be used to produce full-length or truncated versions of the protein with tags for purification and detection .

• CRISPR/Cas9 genome editing: For in vivo studies, CRISPR-mediated knockouts or mutations can be introduced in Xenopus embryos.

• Xenopus oocyte microinjection: For overexpression or localization studies.

What mechanisms regulate FAM73A protein function in Xenopus cell systems?

FAM73A function in Xenopus likely involves regulation at multiple levels:

• Post-translational modifications: The sequence contains multiple predicted phosphorylation sites that may regulate activity, particularly in the C-terminal region around amino acids 500-570 .

• Protein-protein interactions: The coiled-coil domains suggest interaction with other proteins to form functional complexes.

• Subcellular localization: The transmembrane domain suggests membrane localization, potentially at organelles like mitochondria or ER.

• Developmental regulation: Expression levels likely vary throughout development, regulated by tissue-specific transcription factors.

When designing experiments to study these regulatory mechanisms, researchers should consider approaches used for similar proteins in Xenopus systems, such as immunoprecipitation coupled with mass spectrometry to identify interaction partners.

How can recombinant FAM73A be optimally expressed and purified for structural studies?

For optimal expression and purification of recombinant Xenopus laevis FAM73A:

• Expression system selection:

  • E. coli for non-glycosylated protein fragments

  • Insect cells for full-length protein with post-translational modifications

  • Mammalian cells for fully modified protein

• Construct design considerations:

  • Express the protein region 1-570 for full-length studies

  • Include appropriate tags (His, GST) for purification

  • Consider removing transmembrane domains for improved solubility

• Purification protocol:

  • Use affinity chromatography based on the chosen tag

  • Follow with size exclusion chromatography

  • Store in Tris-based buffer with 50% glycerol at -20°C or -80°C

• Quality control:

  • Assess purity by SDS-PAGE

  • Verify identity by mass spectrometry

  • Check folding by circular dichroism

Avoid repeated freeze-thaw cycles, and store working aliquots at 4°C for up to one week to maintain protein integrity .

What is the relationship between FAM73A and genome maintenance mechanisms in Xenopus?

While direct evidence linking FAM73A to genome maintenance in Xenopus is not explicitly detailed in the available sources, contextual analysis suggests potential relationships:

• Temporal correlation: FAM73A is present in unfertilized eggs, which contain machinery for DNA repair and replication .

• Potential interactions: Xenopus egg extracts have been instrumental in studying genome maintenance mechanisms including mismatch repair, non-homologous end joining, interstrand crosslink repair, checkpoint activation, and replication fork stability .

• Research approach: To investigate potential roles of FAM73A in genome maintenance:

  • Perform FAM73A immunodepletion from egg extracts and assess DNA repair capacity

  • Use mass spectrometry to identify FAM73A interaction partners in repair complexes

  • Analyze DNA damage responses in FAM73A-depleted or overexpressing systems

Further research using the Xenopus egg extract system would be valuable for determining if FAM73A plays roles in any of these mechanisms.

How do post-translational modifications affect FAM73A function in Xenopus?

Post-translational modifications (PTMs) likely play crucial roles in regulating FAM73A function:

• Predicted modifications:

  • Phosphorylation sites throughout the protein sequence

  • Potential ubiquitination sites

  • Possible glycosylation sites

• Functional implications:

  • Phosphorylation may regulate protein-protein interactions

  • Ubiquitination could control protein turnover

  • PTMs might affect subcellular localization

• Experimental approaches:

  • Mass spectrometry analysis of endogenous FAM73A

  • Site-directed mutagenesis of key residues

  • In vitro kinase assays

  • Analysis in Xenopus egg extracts with specific kinase inhibitors

The study of PTMs would benefit from the deep proteomics approaches that have been successfully applied to Xenopus egg proteins .

What are the evolutionary implications of FAM73A conservation between Xenopus and mammalian systems?

The conservation of FAM73A between Xenopus and mammals has significant evolutionary implications:

• Functional conservation: Highly conserved proteins typically perform essential cellular functions that have been maintained through evolutionary pressure.

• Structural insights:

  • Conserved domains likely represent functional units

  • Variable regions may indicate species-specific adaptations

• Experimental applications:

  • Xenopus FAM73A can serve as a model for understanding human FAM73A function

  • Cross-species complementation experiments can reveal functional conservation

• Evolutionary analysis approaches:

  • Phylogenetic tree construction using FAM73A sequences from diverse vertebrates

  • Ka/Ks ratio analysis to identify regions under positive or purifying selection

  • Structural prediction comparisons across species

This evolutionary conservation supports using Xenopus as a model system for studying fundamental aspects of FAM73A biology relevant to human health and disease.

What are the optimal conditions for using recombinant FAM73A in in vitro assays?

For optimal use of recombinant Xenopus laevis FAM73A in in vitro assays:

Specific recommendations for different assay types:

• Binding assays: Use freshly thawed protein at 4°C

• Enzymatic assays: Optimize buffer conditions based on predicted activity

• Structural studies: Consider buffer exchange to remove glycerol

• Interaction studies: Add protease inhibitors to prevent degradation

Store working aliquots at 4°C for up to one week to maintain optimal activity .

How can I design effective knockdown/knockout experiments for FAM73A in Xenopus systems?

Designing effective knockdown/knockout experiments for FAM73A in Xenopus:

• Morpholino oligonucleotide approach:

  • Design translation-blocking morpholinos targeting the 5' UTR or start codon region

  • Include control morpholinos (standard control and mismatch)

  • Validate knockdown efficiency by Western blot

  • Inject 1-10 ng morpholino at 1-2 cell stage

• CRISPR/Cas9 genome editing:

  • Design sgRNAs targeting conserved exons (preferably early exons)

  • Prepare Cas9 protein and sgRNA mixtures for injection

  • Validate editing efficiency by T7E1 assay or sequencing

  • Screen F0 embryos for phenotypes and generate F1 lines for stable knockouts

• Rescue experiments:

  • Co-inject mRNA encoding morpholino-resistant or CRISPR-resistant FAM73A

  • Use tagged versions (GFP, mCherry) to confirm expression

  • Include domain mutants to assess structure-function relationships

• Phenotypic analysis:

  • Perform detailed morphological assessment

  • Use molecular markers to assess cellular functions

  • Analyze at multiple developmental stages

What techniques are most effective for studying FAM73A localization in Xenopus cells?

For studying FAM73A localization in Xenopus cells:

• Fluorescent protein fusion approaches:

  • Generate N- or C-terminal GFP/mCherry fusions of FAM73A

  • Express in Xenopus embryos via mRNA injection

  • Analyze by confocal microscopy in live or fixed samples

  • Consider photo-activatable or photo-convertible tags for dynamic studies

• Immunofluorescence techniques:

  • Generate specific antibodies against Xenopus FAM73A

  • Validate antibody specificity using recombinant protein

  • Co-stain with organelle markers (mitochondria, ER, Golgi)

  • Use Xenopus cell lines (XTC, XL177) or primary cultures

• Biochemical fractionation:

  • Prepare subcellular fractions from Xenopus eggs or tissues

  • Analyze FAM73A distribution by Western blot

  • Compare with known organelle markers

  • Consider density gradient approaches for fine resolution

• Electron microscopy approaches:

  • Immunogold labeling for high-resolution localization

  • Correlative light and electron microscopy for context

The transmembrane domain in the FAM73A sequence suggests it may localize to cellular membranes, potentially at organelles like mitochondria or ER .

How can I identify and validate FAM73A interaction partners in Xenopus systems?

To identify and validate FAM73A interaction partners in Xenopus:

• Co-immunoprecipitation approaches:

  • Generate tagged versions of FAM73A (FLAG, HA, His)

  • Express in Xenopus eggs or embryos

  • Perform pull-downs followed by Western blot or mass spectrometry

  • Validate with reciprocal pull-downs

• Proximity labeling techniques:

  • Generate BioID or APEX2 fusions with FAM73A

  • Express in Xenopus systems and provide biotin

  • Identify labeled proteins by streptavidin pull-down and mass spectrometry

  • Validate top candidates by co-localization studies

• Yeast two-hybrid screening:

  • Use FAM73A domains as bait against Xenopus cDNA libraries

  • Verify interactions in mammalian cells

  • Confirm in Xenopus using co-IP or FRET approaches

• In vitro validation:

  • Express and purify recombinant proteins

  • Perform direct binding assays

  • Use surface plasmon resonance or isothermal titration calorimetry for quantitative analyses

The coiled-coil domains in FAM73A suggest it likely forms protein-protein interactions, potentially as part of larger complexes .

What approaches can be used to assess FAM73A function in Xenopus egg extract systems?

Assessing FAM73A function in Xenopus egg extract systems:

• Immunodepletion studies:

  • Deplete endogenous FAM73A using specific antibodies

  • Add back recombinant wild-type or mutant FAM73A

  • Assess effects on specific biochemical pathways

  • Compare to mock-depleted controls

• Xenopus egg extract preparation:

  • Prepare conventional unfractionated extract by crushing mature Xenopus eggs

  • Optimize extract conditions to maintain FAM73A activity

  • Use fresh extract or flash-freeze in liquid nitrogen for storage

• Functional assays in extracts:

  • DNA replication assays using plasmid templates

  • DNA repair assays (similar to those used for studying genome maintenance)

  • Protein modification assays (phosphorylation, ubiquitination)

  • Membrane association studies

• Biochemical analysis:

  • Track FAM73A modifications during extract incubation

  • Monitor protein complex formation using size exclusion chromatography

  • Assess membrane association or recruitment

Xenopus egg extracts provide a powerful system for studying protein function as they contain all factors required to efficiently perform complex cellular processes outside a cell .

How can I resolve issues with recombinant FAM73A solubility and stability?

Troubleshooting recombinant FAM73A solubility and stability issues:

For storage, a Tris-based buffer with 50% glycerol at pH 8.0 is recommended, and working aliquots should be kept at 4°C for up to one week . The addition of 6% Trehalose to the storage buffer can further enhance stability .

How do I interpret contradictory results between in vitro and in vivo FAM73A studies?

When faced with contradictory results between in vitro and in vivo FAM73A studies:

• Systematic comparison:

  • Create a detailed table of experimental conditions

  • Identify key differences in protein concentration, buffer conditions, and assay endpoints

  • Consider time-scale differences between systems

• Biological context considerations:

  • In vivo studies include regulatory factors that may be missing in vitro

  • Post-translational modifications may differ between systems

  • Protein localization constraints exist in vivo but not in vitro

• Reconciliation approaches:

  • Develop intermediate systems (e.g., Xenopus egg extracts) that bridge in vitro and in vivo

  • Isolate subcellular compartments for more contextual in vitro studies

  • Create reconstituted systems with defined components

• Interpretation framework:

  • In vitro studies reveal biochemical potential

  • In vivo studies show physiological relevance

  • Both approaches provide valid but different aspects of protein function

Xenopus egg extract systems provide a valuable intermediate between purely in vitro and in vivo approaches, as they maintain cellular context while allowing experimental manipulation .

What statistical approaches are appropriate for analyzing FAM73A expression or function data?

Appropriate statistical approaches for analyzing FAM73A data:

• Expression analysis:

  • Normalization methods: GAPDH, β-actin, or global normalization for proteomics

  • Statistical tests: t-test for two-condition comparisons, ANOVA for multiple conditions

  • Multiple testing correction: Benjamini-Hochberg for false discovery rate control

  • Visualization: Box plots, violin plots for distribution comparison

• Functional assays:

  • Replicate requirements: Minimum 3 biological replicates with 2-3 technical replicates each

  • Power analysis: Determine sample size needed for detecting effect sizes of interest

  • Statistical tests: Paired tests for before/after comparisons, repeated measures ANOVA for time series

• Protein-protein interaction analysis:

  • Enrichment calculations: Compare to appropriate negative controls

  • Significance testing: Permutation tests for interaction networks

  • Multiple hypothesis testing: Control for family-wise error rate

• Deep proteomics approaches:

  • Specialized statistical methods similar to those used in the analysis of >11,000 proteins from Xenopus eggs with 99% confidence and approximately two-fold precision for abundance estimation

When reporting results, include exact p-values, confidence intervals, and effect size measurements.

How can I address non-specific effects when studying FAM73A in Xenopus systems?

Addressing non-specific effects in FAM73A studies:

• Experimental design controls:

  • Include scrambled/mismatched controls for knockdown studies

  • Use catalytically inactive mutants as controls for overexpression

  • Perform dose-response studies to identify specific vs. non-specific effects

  • Include appropriate negative controls (unrelated proteins of similar size/structure)

• Validation approaches:

  • Use multiple independent methods to confirm findings

  • Perform rescue experiments with resistant constructs

  • Create structure-function correlations through domain mutations

  • Validate in different cellular contexts or developmental stages

• Specificity confirmation:

  • For antibodies: Pre-absorb with recombinant protein

  • For morpholinos/CRISPR: Confirm target reduction by Western blot

  • For overexpression: Verify physiological levels with quantitative methods

• Cross-species validation:

  • Test if human FAM73A can rescue Xenopus FAM73A depletion

  • Compare phenotypes with those in other model organisms

The rigorous experimental systems established for Xenopus egg extracts provide good models for implementing these controls .

What are the best practices for comparing FAM73A function across different developmental stages in Xenopus?

Best practices for comparing FAM73A function across Xenopus developmental stages:

• Experimental design considerations:

  • Use a developmental series with standardized staging

  • Maintain consistent experimental conditions across stages

  • Include stage-appropriate controls

  • Consider maternal vs. zygotic contributions at early stages

• Quantification approaches:

  • Normalize protein expression to stage-specific reference proteins

  • Use ratiometric measurements for functional assays

  • Account for differences in cell number/protein content between stages

  • Consider tissue-specific expression patterns at later stages

• Analytical framework:

  • Distinguish between absolute and relative changes

  • Consider allometric scaling for size-dependent processes

  • Use developmental trajectory analysis rather than pairwise comparisons

  • Implement mixed-effects models to account for batch variation

• Visualization and reporting:

  • Plot data against standardized developmental time points

  • Include embryo images for phenotypic analyses

  • Present results in the context of known developmental events

  • Report stage-specific differences in protein localization or modification

The deep proteomics approaches that have been applied to Xenopus eggs could be extended across developmental stages for comprehensive analysis of FAM73A expression and modification patterns .

What are the emerging research directions for FAM73A protein studies in Xenopus?

Emerging research directions for FAM73A protein studies in Xenopus include:

• Systems biology approaches:

  • Integration of FAM73A into protein interaction networks

  • Computational modeling of FAM73A function

  • Multi-omics integration (proteomics, transcriptomics, metabolomics)

• Comparative evolutionary studies:

  • Analysis across Xenopus species (laevis, tropicalis)

  • Comparison with mammalian FAM73A function

  • Identification of conserved functional motifs

• Advanced imaging techniques:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging for dynamic studies

  • Correlative light and electron microscopy

• Disease relevance:

  • Modeling human FAM73A mutations in Xenopus

  • Investigating roles in cellular pathways relevant to disease

  • Drug screening in Xenopus egg extract systems