Recombinant Mouse Ankyrin repeat domain-containing protein 46 (Ankrd46)

What is the basic structure of mouse Ankrd46 protein?

Mouse Ankrd46 is a protein characterized by multiple ankyrin repeat domains. These ankyrin domains form a β-hairpin–α-helix–loop–α-helix (β2α2) secondary structure that functions in protein-protein interactions across various cellular processes . The protein contains multiple ankyrin repeats that are essential for its biological function. The mouse Ankrd46 is orthologous to human ANKRD46, with high sequence identity between species .

What are the known domains and functional regions of Ankrd46?

Ankrd46 contains multiple ankyrin repeat domains, which are approximately 33-amino acid motifs that create protein-binding interfaces. These domains are arranged in tandem arrays and contribute to the protein's three-dimensional structure, allowing it to participate in protein-protein interactions. Additionally, mouse Ankrd46a (a zebrafish ortholog) is predicted to be located in the membrane according to comparative studies . The protein functions in various cellular processes through its protein-protein interaction capabilities mediated by these ankyrin domains .

How conserved is Ankrd46 across species?

Ankrd46 shows significant conservation across mammalian species. Human ANKRD46 protein has orthologues in mouse and rat with approximately 98% sequence identity in these species . This high degree of evolutionary conservation suggests important biological functions. Zebrafish has an orthologous gene called ankrd46a, which is located on chromosome 21 . This conservation across vertebrate species indicates functional importance throughout evolutionary history.

What is the expression pattern of Ankrd46 in mouse tissues?

While the search results don't provide comprehensive tissue expression data specifically for mouse Ankrd46, comparative studies suggest that it may have a pattern similar to human ANKRD46. Studies utilizing mouse models have shown that Ankrd46 may be expressed in various tissues, as evidenced by phenotypic changes in multiple systems when the gene is disrupted . Mouse models with Ankrd46 mutations have demonstrated abnormal vitreous body morphology and increased monocyte cell numbers, suggesting expression in ocular tissues and hematopoietic systems .

How do I design experiments to study the temporal expression of Ankrd46 during development?

To study temporal expression of Ankrd46 during development:

• RNA expression analysis:

  • Isolate RNA from mouse tissues at different developmental stages

  • Perform RT-qPCR using validated primers specific for Ankrd46

  • Follow MIQE guidelines for qPCR experimental design to ensure reliability

  • Design primers that span exon-exon junctions to avoid genomic DNA amplification

• Protein expression analysis:

  • Collect tissue samples from multiple developmental timepoints

  • Perform Western blotting using validated anti-Ankrd46 antibodies

  • Include appropriate loading controls and quantification methods

  • Consider using immunohistochemistry to determine spatial localization

• Single-cell analysis approach:

  • Implement single-cell RNA sequencing to detect cell-type specific expression patterns

  • Analyze data using computational tools to track expression changes during differentiation

  • Compare expression patterns with developmental markers

What expression systems are most effective for producing recombinant mouse Ankrd46?

Based on established protocols for similar ankyrin repeat proteins, the most effective expression systems for recombinant mouse Ankrd46 production include:

• Bacterial expression systems (E. coli):

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

  • Considerations: May require optimization of codon usage for mammalian proteins

  • Best for: Protein fragments, domains, or full-length protein without post-translational modifications

  • Expression tags: His-tags are commonly used for purification purposes

• Insect cell expression (Baculovirus):

  • Advantages: Better folding of complex proteins, some post-translational modifications

  • Considerations: More time-consuming than bacterial systems

  • Best for: Full-length protein requiring proper folding

• Mammalian cell expression (CHO, HEK293):

  • Advantages: Native-like post-translational modifications, proper folding

  • Considerations: Lower yields, higher cost

  • Best for: Studies requiring functional protein with authentic modifications

The choice depends on experimental requirements for protein function, purity, and yield.

What purification strategies yield the highest purity for recombinant Ankrd46?

For high-purity recombinant Ankrd46, a multi-step purification strategy is recommended:

• Affinity chromatography:

  • For His-tagged constructs: Ni-NTA or IMAC purification

  • Optimize imidazole concentration in washing steps to reduce non-specific binding

  • Consider on-column refolding if protein is expressed in inclusion bodies

• Ion exchange chromatography:

  • As a secondary purification step based on the protein's isoelectric point

  • Helps remove contaminants with similar affinity but different charge properties

• Size exclusion chromatography:

  • Final polishing step to separate monomeric protein from aggregates

  • Also useful for buffer exchange into storage buffer

• Quality control:

  • Assess purity by SDS-PAGE (≥95% purity is typically desired)

  • Verify identity by Western blot or mass spectrometry

  • Test for endotoxin contamination using LAL assay (<0.1 EU/μg is standard for research applications)

How should recombinant Ankrd46 be stored to maintain stability and activity?

Based on protocols for similar proteins, optimal storage conditions for recombinant Ankrd46 include:

• Short-term storage (1-2 weeks):

  • Store at 4°C in appropriate buffer with protease inhibitors

  • Avoid repeated freeze-thaw cycles

• Long-term storage:

  • Aliquot protein to avoid repeated freeze-thaw cycles

  • Store at -20°C or -80°C in buffer containing:

    • 50% glycerol to prevent freezing damage

    • PBS with mild preservative (e.g., 0.01% thimerosal, pH 7.3)

    • Optional: 0.1% BSA as carrier protein to prevent adsorption to tubes

• Lyophilization (for extended stability):

  • Lyophilize from a filtered solution

  • Store lyophilized protein at -20°C

  • Reconstitute in sterile water or appropriate buffer

• Stability monitoring:

  • Perform periodic activity assays to confirm protein functionality

  • Check for degradation using SDS-PAGE before critical experiments

What are the most reliable methods to assess Ankrd46 protein-protein interactions?

Several robust methods can be employed to characterize Ankrd46 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-Ankrd46 antibodies to pull down protein complexes

  • Identify interacting partners by mass spectrometry

  • Confirm interactions with Western blotting

  • Include appropriate controls (IgG, lysate input)

• Proximity-based labeling techniques:

  • BioID or TurboID fusion constructs to identify proximal proteins

  • APEX2 labeling for temporal control of labeling reactions

  • Analyze results with mass spectrometry

• Yeast two-hybrid screening:

  • Use Ankrd46 (or specific domains) as bait

  • Screen against mouse cDNA libraries

  • Validate interactions with orthogonal methods

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

  • Quantitative measurement of binding kinetics and affinity

  • Requires purified recombinant proteins

  • Can determine association/dissociation rates

• Fluorescence techniques:

  • Förster resonance energy transfer (FRET)

  • Fluorescence correlation spectroscopy (FCS)

  • Bimolecular fluorescence complementation (BiFC)

These approaches provide complementary information about the specificity, strength, and context of interactions.

How can I design experiments to study the role of Ankrd46 in specific cellular processes?

To study Ankrd46's role in cellular processes, consider these experimental approaches:

• Loss-of-function studies:

  • CRISPR/Cas9-mediated gene knockout

  • siRNA or shRNA knockdown for transient depletion

  • Use multiple guide RNAs or siRNAs to control for off-target effects

  • Include rescue experiments with wild-type Ankrd46 to confirm specificity

• Gain-of-function studies:

  • Overexpression of wild-type Ankrd46

  • Creation of domain-specific mutants to identify functional regions

  • Use inducible expression systems for temporal control

• Localization studies:

  • Generate fluorescent protein fusions

  • Perform live cell imaging to track dynamics

  • Co-localization with organelle markers

• Phenotypic assays:

  • Based on mouse knockout phenotypes , consider:

    • Measuring cell size and proliferation (decreased body size phenotype)

    • Quantifying monocyte numbers by flow cytometry (increased monocyte cell number phenotype)

    • Examining eye morphology (abnormal vitreous body morphology phenotype)

• Single-cell analysis:

  • Transcriptomic profiling after Ankrd46 manipulation

  • Pathway analysis to identify affected cellular processes

Importantly, experimental design should follow established guidelines such as proper controls, statistical power analysis, and randomization to ensure reliable results .

What functional assays can determine if recombinant Ankrd46 retains native activity?

To assess whether recombinant Ankrd46 retains its native functional activity:

• Protein-binding assays:

  • Pull-down assays with known or predicted interaction partners

  • SPR or BLI to measure binding kinetics

  • Compare binding properties with native protein when possible

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure composition

  • Thermal stability assays to assess proper folding

  • Limited proteolysis to evaluate domain organization

  • Techniques similar to those used for annexin A11 , which showed distinct CD spectral changes upon calcium binding

• Cellular activity assays:

  • Competition assays with endogenous protein

  • Rescue experiments in Ankrd46-depleted cells

  • Membrane localization assessment, as Ankrd46 is predicted to associate with membranes

• Post-translational modification analysis:

  • Phosphorylation state assessment (if known to be regulated by phosphorylation)

  • Western blotting with modification-specific antibodies

• Comparison with non-recombinant controls:

  • Side-by-side assays with native protein when available

  • Use of multiple recombinant protein batches to ensure consistency

What is the evidence linking Ankrd46 to human diseases?

Current evidence linking Ankrd46 to human diseases includes:

• Alcohol Use Disorder (AUD):

  • A whole exome sequencing study identified ANKRD46 as having protein-truncating variants associated with loss of function that differed in frequency between AUD probands and controls

  • The study found that ANKRD46 had an odds ratio (OR) of 176.1 (p = 2.01 × 10⁻⁴) in AUD patients

  • This suggests ANKRD46 may play a role in neurological pathways related to addiction

• Atrioventricular conduction:

  • ANKRD46 has been associated with genomic predictors of atrioventricular conduction based on electronic medical records

  • This implies potential involvement in cardiac electrical signaling

• Other potential associations:

  • Ankyrin repeat domain proteins as a family have been implicated in various diseases

  • For example, ANKRD13D has been identified as a potential target in renal cell carcinoma

  • ANKRD46's role in protein-protein interactions suggests it could influence multiple cellular pathways relevant to disease

How can mouse models help understand Ankrd46 function in disease contexts?

Mouse models provide valuable insights into Ankrd46 function in disease contexts through:

• Existing knockout phenotypes:

  • Ankrd46 em1(IMPC)Mbp/Ankrd46 em1(IMPC)Mbp mice show:

    • Abnormal vitreous body morphology

    • Increased monocyte cell number

  • Ankrd46 em1Tmg/Ankrd46 em1Tmg mice display:

    • Decreased body size

  • These phenotypes suggest roles in ocular development, immune function, and growth regulation

• Disease model development:

  • Conditional knockout models can target Ankrd46 deletion to specific tissues

  • Inducible systems allow temporal control to distinguish developmental vs. adult functions

  • Humanized mouse models could incorporate human ANKRD46 variants identified in diseases

• Experimental approaches:

  • Behavioral testing for AUD-related phenotypes (based on human association)

  • Cardiac conduction studies (ECG, optical mapping)

  • Immune cell profiling (flow cytometry, single-cell analysis)

  • Cross with disease model mice to assess genetic interactions

• Translational applications:

  • Drug screening on Ankrd46-deficient cells or mice

  • Biomarker development based on altered pathways

  • Target validation for therapeutic development

When designing these studies, researchers should consider:

• Use of both sexes to account for potential sex differences

• Multiple genetic backgrounds to control for strain-specific effects

• Appropriate age ranges based on disease onset

• Rigorous statistical design with adequate sample sizes

What methodological approaches can assess Ankrd46 as a therapeutic target?

To evaluate Ankrd46 as a potential therapeutic target, researchers should consider these methodological approaches:

• Target validation:

  • CRISPR/Cas9 knockout in disease-relevant cell lines

  • Conditional knockout in adult mice to avoid developmental effects

  • RNA interference for acute, tissue-specific knockdown

  • Correlation of expression levels with disease progression in patient samples

• Functional screening:

  • High-throughput screening for small molecules that modulate Ankrd46 interactions

  • Fragment-based drug discovery targeting specific protein domains

  • Peptide-based inhibitors of key protein-protein interactions

  • Structure-based drug design (if crystal structure becomes available)

• Mechanism of action studies:

  • Identification of critical binding partners in disease contexts

  • Mapping of interaction interfaces using mutagenesis

  • Characterization of downstream signaling pathways

  • Phosphoproteomics to identify altered signaling networks

• Therapeutic delivery approaches:

  • For protein replacement: recombinant protein delivery systems

  • For gene therapy: viral vector optimization for tissue-specific expression

  • For inhibition: antisense oligonucleotides or siRNA delivery strategies

  • Evaluation of tissue penetration and pharmacokinetics

• Safety assessment:

  • Off-target effect analysis using transcriptomics and proteomics

  • Toxicity studies in diverse cell types

  • Evaluation of compensatory mechanisms upon target inhibition

These approaches should be implemented with careful experimental design, including appropriate controls and statistical power calculations to ensure reliable and reproducible results.

How do post-translational modifications affect Ankrd46 function and interactions?

While specific information about post-translational modifications (PTMs) of Ankrd46 is limited in the provided search results, researchers can investigate this important aspect through:

• Identification of PTMs:

  • Mass spectrometry-based proteomics to map PTM sites

  • Comparison of PTMs between recombinant and endogenous protein

  • Analysis of PTM dynamics under different cellular conditions

  • Examination of conservation of potential modification sites across species

• Functional impact assessment:

  • Site-directed mutagenesis of predicted PTM sites (phosphorylation, ubiquitination, etc.)

  • Generation of phosphomimetic or non-phosphorylatable mutants

  • Comparison of binding properties before and after inducing specific modifications

  • Analysis of protein stability and half-life with modified or unmodified protein

• Regulatory enzymes:

  • Identification of kinases, phosphatases, or other enzymes that modify Ankrd46

  • Co-expression studies to assess enzyme-substrate relationships

  • Inhibitor studies to determine functional consequences of specific modifications

• Structural consequences:

  • Molecular dynamics simulations to predict conformational changes

  • Biophysical methods (CD, fluorescence) to detect structural alterations

  • NMR studies of domain flexibility before and after modification

This research direction may be particularly important given that many ankyrin repeat proteins are regulated by PTMs that affect their binding properties and cellular functions.

What is the relationship between Ankrd46 expression and immune cell function?

The relationship between Ankrd46 and immune cell function represents an intriguing research area, particularly given the phenotype of increased monocyte numbers observed in knockout mice :

• Expression profiling:

  • Single-cell RNA sequencing of immune cell populations

  • Flow cytometry analysis of Ankrd46 protein levels across immune cell subtypes

  • Examination of expression changes during immune cell differentiation and activation

• Functional studies in immune cells:

  • CRISPR/Cas9-mediated knockout in primary immune cells or cell lines

  • Assessment of proliferation, differentiation, and cytokine production

  • Chemotaxis and migration assays to evaluate cellular motility

  • Phagocytosis and other functional assays for myeloid cells

• Signaling pathway analysis:

  • Phosphoproteomic analysis of signaling changes in Ankrd46-deficient immune cells

  • Investigation of receptor-mediated signaling (e.g., cytokine receptors, TLRs)

  • Analysis of transcription factor activation downstream of Ankrd46

• In vivo immune challenges:

  • Response to infectious agents in Ankrd46-deficient mice

  • Autoimmune disease models to assess regulatory functions

  • Bone marrow transplantation to distinguish intrinsic vs. extrinsic effects

• Clinical correlations:

  • Analysis of ANKRD46 expression in patient immune cells in various disease states

  • Correlation with inflammatory markers or disease severity

  • Genetic association studies in immune-related disorders

These studies would help clarify whether Ankrd46 has direct functions in immune cells or whether the observed monocyte phenotype is secondary to other physiological changes.

How does alternative splicing regulate Ankrd46 expression and function in different tissues?

Alternative splicing is a key regulatory mechanism for Ankrd46, as indicated by the existence of multiple transcript variants . To investigate this process:

• Comprehensive transcript mapping:

  • RNA-seq analysis across tissues and developmental stages

  • Targeted amplification of alternative exons

  • 3' and 5' RACE to identify all transcript isoforms

  • Quantification of isoform abundance in different contexts

• Isoform-specific functions:

  • Generation of isoform-specific expression constructs

  • Rescue experiments with different isoforms in knockout models

  • Domain-specific analysis of protein-protein interactions

  • Subcellular localization studies for each isoform

• Splicing regulation:

  • Identification of splicing enhancers and silencers within the gene

  • Investigation of tissue-specific splicing factors that interact with these elements

  • Minigene assays to test specific regulatory sequences

  • CLIP-seq to identify RNA-binding proteins that regulate Ankrd46 splicing

• Evolutionary conservation:

  • Comparative analysis of splicing patterns across species

  • Identification of conserved vs. species-specific isoforms

  • Analysis of selection pressure on alternatively spliced exons

• Disease relevance:

  • Analysis of splicing changes in disease states

  • Investigation of splicing-disrupting mutations in patient samples

  • Therapeutic approaches targeting specific splice variants

This research area is particularly important as alternative splicing can dramatically alter protein function, creating isoforms with distinct or even opposing activities from the same gene.

What are common challenges in detecting endogenous Ankrd46 and how can they be overcome?

Detecting endogenous Ankrd46 can present several challenges:

• Antibody specificity issues:

  • Challenge: Cross-reactivity with other ankyrin repeat proteins

  • Solution: Validate antibodies using knockout controls and multiple antibodies targeting different epitopes

  • Method: Compare commercial antibodies through Western blot, immunoprecipitation, and immunocytochemistry applications

• Low expression levels:

  • Challenge: Weak signal in certain tissues or cell types

  • Solution: Use signal amplification methods or enrichment strategies

  • Method: Consider proximity ligation assays, tyramide signal amplification, or immunoprecipitation before detection

• Isoform complexity:

  • Challenge: Multiple splice variants complicating interpretation

  • Solution: Design isoform-specific detection reagents

  • Method: Use primers spanning unique exon junctions for RT-qPCR, or antibodies targeting isoform-specific regions

• Protein extraction difficulties:

  • Challenge: Membrane association may require specialized extraction methods

  • Solution: Optimize lysis buffers with appropriate detergents

  • Method: Compare different extraction protocols (RIPA, NP-40, Triton X-100, etc.) for efficiency

• Post-translational modifications:

  • Challenge: Modifications may mask epitopes or alter migration

  • Solution: Use multiple detection methods and consider modification-specific antibodies

  • Method: Treat samples with phosphatases or other enzymes to remove modifications when needed

Following MIQE guidelines for qPCR and similar best practices for protein detection can help ensure reliable results.

How should experiments be designed to analyze Ankrd46 gene expression changes reliably?

For reliable analysis of Ankrd46 gene expression:

• RT-qPCR optimization:

  • Follow MIQE guidelines for experimental design and reporting

  • Validate primer efficiency using standard curves (90-110% efficiency ideal)

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Use multiple primer sets targeting different regions of the transcript

  • Include no-RT controls to detect genomic DNA contamination

• Reference gene selection:

  • Critical point: Common reference genes like GAPDH and ACTB have been shown to vary considerably under different experimental conditions

  • Test multiple reference genes for stability in your specific experimental system

  • Use reference gene validation tools (GeNorm, NormFinder)

  • Consider using geometric mean of multiple validated reference genes

• Sample preparation:

  • Ensure high RNA quality (RIN > 8) for reliable results

  • Complete removal of RNA from cDNA samples is essential for accurate quantification

  • Standardize RNA input amounts across all samples

  • Process all samples in parallel when possible

• Experimental design considerations:

  • Include biological replicates (minimum n=3, preferably more)

  • Include technical replicates to assess method precision

  • Randomize sample processing to avoid batch effects

  • Use power analysis to determine appropriate sample sizes

• Data analysis:

  • Apply appropriate normalization methods

  • Use statistics appropriate for the experimental design

  • Report both biological and technical variability

  • Consider advanced analyses for complex designs (e.g., mixed effects models)

What quality control measures are essential when working with recombinant Ankrd46?

Essential quality control measures for recombinant Ankrd46 include:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (≥95% purity is standard)

  • Densitometric analysis for quantification

  • Size exclusion chromatography to detect aggregates

  • Mass spectrometry for precise molecular weight determination and contaminant identification

• Identity confirmation:

  • Western blotting with specific antibodies

  • Mass spectrometry peptide mapping

  • N-terminal sequencing to confirm correct processing

  • Verification of any affinity tags (His-tag, etc.)

• Functional validation:

  • Binding assays with known interaction partners

  • Structural characterization (CD spectroscopy, thermal stability)

  • Comparison with positive control batches when available

  • Activity assays appropriate to the protein's function

• Contaminant testing:

  • Endotoxin testing using LAL assay (<0.1 EU/μg protein is standard)

  • Host cell protein analysis by ELISA or mass spectrometry

  • DNA contamination assessment

  • Sterility testing if required for cell-based applications

• Stability monitoring:

  • Accelerated stability studies under various conditions

  • Real-time stability monitoring during storage

  • Freeze-thaw stability assessment

  • Assessment of activity retention over time

• Batch-to-batch consistency:

  • Standardized production methods

  • Reference standards for comparison

  • Consistent quality control metrics across batches

  • Documentation of production parameters

These measures ensure that experimental results are reliable and reproducible when working with recombinant Ankrd46 protein.