Recombinant Mouse EF-hand domain-containing protein KIAA0494 (Kiaa0494)

How does mouse KIAA0494 compare structurally to its human ortholog?

When comparing mouse KIAA0494 to the human ortholog, several structural similarities and differences emerge. The human KIAA0494 protein (from the available recombinant constructs) shows approximately 87% sequence identity with its mouse counterpart . Both proteins contain the core EF-hand calcium-binding domains, but exhibit species-specific differences particularly in:

• The N-terminal region where the mouse protein contains several unique residues

• The central domain where several calcium-binding motifs show subtle variations in key amino acids

• The C-terminal region where post-translational modification sites may differ

Recombinant expression systems for both species typically use mammalian hosts such as HEK293T cells to ensure proper folding and post-translational modifications . This conservation suggests functional importance across species while allowing for species-specific regulatory mechanisms.

What are the optimal conditions for expressing recombinant mouse KIAA0494 protein?

Based on established protocols, the optimal conditions for expressing recombinant mouse KIAA0494 include:

• Expression System: Mammalian expression systems, particularly HEK-293 cells, have proven most effective for maintaining proper folding and post-translational modifications of mouse KIAA0494 . Bacterial systems often yield misfolded protein due to the complex structure of the EF-hand domains.

• Transfection Protocol: Transient transfection using lipid-based reagents with 5μg ORF cDNA plasmid per 10-cm dish of cells yields optimal expression . Cells should be cultured for 48 hours post-transfection before harvesting.

• Cell Lysis Conditions: Modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, proteinase inhibitor cocktail, 1mM PMSF, and 1mM Na₃VO₄) effectively extracts the protein while preserving structural integrity .

• Purification Strategy: For His-tagged constructs, one-step affinity chromatography using nickel or cobalt resins under native conditions works effectively, with elution using 250mM imidazole .

These conditions consistently yield protein preparations with >80% purity as determined by SDS-PAGE analysis.

What antibodies are optimal for detecting mouse KIAA0494 in different experimental applications?

The selection of appropriate antibodies for mouse KIAA0494 detection depends on the specific application:

When working with tagged recombinant proteins, anti-tag antibodies provide excellent specificity. For mouse KIAA0494 with His-tag, anti-His antibodies work well for detection, while for DDK/FLAG-tagged constructs, the OTI4C5 clone has shown high specificity .

How can I investigate the calcium-binding properties of mouse KIAA0494 protein?

Investigating calcium-binding properties of mouse KIAA0494 requires multiple complementary approaches:

• Isothermal Titration Calorimetry (ITC): This technique directly measures calcium binding thermodynamics. Using purified recombinant KIAA0494 (>90% purity), titrate CaCl₂ solutions (typically 1-10mM) into a protein solution (10-50μM). The resulting thermograms will provide binding constants, stoichiometry, and thermodynamic parameters.

• Circular Dichroism (CD) Spectroscopy: CD spectroscopy can detect calcium-induced conformational changes. Compare spectra of the protein (0.1-0.5 mg/mL) in calcium-free buffer (with EGTA) versus calcium-saturated conditions (1-5mM CaCl₂).

• Fluorescence Spectroscopy: If the protein contains tryptophan residues near calcium-binding sites, intrinsic fluorescence changes upon calcium binding can be monitored. Alternatively, use calcium-sensitive fluorescent dyes like Fura-2 to monitor calcium binding indirectly.

• Mutagenesis Studies: Create point mutations in the predicted EF-hand domains (particularly the DxDxDG motifs) and compare calcium binding properties with wild-type protein using the methods above. This targeted approach can identify which specific EF-hand domains are functional.

These methodologies should be performed with recombinant mouse KIAA0494 purified under native conditions to preserve the structural integrity of the calcium-binding domains .

What approaches can resolve contradictory data regarding KIAA0494 subcellular localization?

When faced with contradictory data regarding KIAA0494 subcellular localization, implement this systematic troubleshooting strategy:

• Multiple Detection Methods: Compare results using both tag-based detection (anti-His or anti-DDK antibodies) and protein-specific antibodies . Discrepancies might arise from tag interference with localization signals.

• Cell Type Considerations: Examine localization in multiple cell types, as KIAA0494 may exhibit cell-type specific localization patterns. Compare results from primary cells with those from cell lines like HEK293T used for recombinant expression .

• Fractionation Validation: Perform subcellular fractionation followed by Western blotting to biochemically validate microscopy-based localization data. This approach provides quantitative distribution across cellular compartments.

• Calcium Dependency: Assess localization under varying calcium conditions (calcium-free, physiological calcium, high calcium) as EF-hand proteins often undergo calcium-dependent translocation.

• Fusion Constructs Analysis: Create multiple fusion constructs with different tags (N-terminal versus C-terminal) to determine if tag position affects localization signals.

To systematically document these findings, create a comprehensive table correlating detection method, cell type, calcium conditions, and observed localization patterns to identify consistent patterns amid seemingly contradictory data.

What experimental approaches can elucidate the physiological role of mouse KIAA0494 in calcium signaling pathways?

To investigate the physiological role of mouse KIAA0494 in calcium signaling pathways, implement these complementary approaches:

• Protein Interaction Studies: Use recombinant His-tagged or DDK-tagged KIAA0494 protein for pull-down assays followed by mass spectrometry to identify interacting partners . This approach can place KIAA0494 within specific calcium signaling networks.

• Calcium Imaging in Manipulated Cells: Perform calcium imaging experiments in cells where KIAA0494 has been:

  • Overexpressed using transient transfection approaches

  • Knocked down using siRNA/shRNA

  • Knocked out using CRISPR-Cas9

  Compare calcium responses to various stimuli across these conditions using ratiometric calcium indicators.

• Phosphorylation Analysis: Investigate whether KIAA0494 is phosphorylated in a calcium-dependent manner by performing phosphoproteomic analysis of cells under resting and stimulated conditions. Recombinant protein can serve as a substrate in in vitro kinase assays.

• Domain Function Analysis: Create truncation constructs removing specific EF-hand domains to determine which regions are essential for function. Express these as recombinant proteins and assess their impact on calcium signaling when introduced into cells .

• Tissue Expression Profiling: Perform quantitative analysis of KIAA0494 expression across tissues using antibodies validated for specificity to identify physiologically relevant systems for deeper investigation.

These approaches collectively provide a comprehensive framework for elucidating KIAA0494's role in calcium homeostasis and signaling.

How can I design experiments to investigate potential protein-protein interactions of mouse KIAA0494?

Designing rigorous experiments to investigate KIAA0494 protein-protein interactions requires a multi-faceted approach:

• Recombinant Protein-Based Pull-Down Assays:

  • Immobilize purified His-tagged KIAA0494 on nickel resin

  • Incubate with tissue or cell lysates under various calcium concentrations (0, 1, 10 μM free Ca²⁺)

  • Elute and analyze binding partners by mass spectrometry

  • Confirm key interactions with Western blotting

• Co-Immunoprecipitation Studies:

  • Express DDK-tagged KIAA0494 in HEK293T cells

  • Immunoprecipitate using anti-DDK antibodies

  • Compare interactomes under resting conditions versus calcium mobilization

  • Validate identified interactions reciprocally

• Proximity Labeling Techniques:

  • Generate BioID or APEX2 fusion constructs with KIAA0494

  • Express in relevant cell types and activate labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures transient and weak interactions in native cellular contexts

• Fluorescence Resonance Energy Transfer (FRET):

  • Create fluorescent protein fusions with KIAA0494 and candidate interactors

  • Measure FRET in live cells under various calcium conditions

  • This provides spatial and temporal resolution of interactions

• Yeast Two-Hybrid Screening:

  • Use KIAA0494 as bait to screen mouse cDNA libraries

  • Validate positive hits using the above methods

  • Consider calcium dependency through modified Y2H systems

These methods should be implemented with appropriate controls, including calcium chelators (EGTA) and calcium ionophores to understand calcium-dependency of identified interactions.

What analytical techniques can resolve aggregation issues with recombinant mouse KIAA0494 protein?

When encountering aggregation issues with recombinant mouse KIAA0494, implement this analytical and troubleshooting framework:

• Dynamic Light Scattering (DLS): Analyze protein preparations (0.5-1 mg/mL) to determine size distribution profiles. Monomeric KIAA0494 should show a narrow peak with low polydispersity index (<0.2), while aggregated samples will show larger hydrodynamic radii and higher polydispersity.

• Size Exclusion Chromatography (SEC): Run purified KIAA0494 on analytical SEC columns (Superdex 200 or similar) to separate monomeric protein from higher-order aggregates. Collect fractions for subsequent functional testing to determine which forms retain activity.

• Buffer Optimization Matrix:

  Buffer Component	Range to Test	Effect on Aggregation
  pH	6.5-8.5 in 0.5 increments	Affects surface charge distribution
  NaCl	50-500 mM	Shields electrostatic interactions
  Calcium	0-5 mM	May stabilize EF-hand domains
  Glycerol	0-10%	Stabilizes hydrophobic interactions
  Detergents	0.01-0.1% non-ionic	Disrupts hydrophobic aggregation

• Thermal Shift Assays: Perform differential scanning fluorimetry with SYPRO Orange to identify buffer conditions that maximize thermal stability, which often correlates with reduced aggregation propensity.

• Expression Strategy Modification: Consider:

  • Lowering expression temperature to 30°C for 72 hours instead of 37°C for 48 hours

  • Co-expressing molecular chaperones

  • Creating truncation constructs that remove aggregation-prone regions

When preparing recombinant KIAA0494 for biochemical studies, centrifuge samples at 100,000×g for 30 minutes to remove any residual aggregates before functional assays.

What is the significance of KIAA0494's conserved domains for designing gene editing experiments?

When designing gene editing experiments targeting KIAA0494, the protein's conserved domains provide critical guidance for experimental design:

• EF-hand Domain Targeting Strategy: The EF-hand calcium-binding domains are highly conserved functional units of KIAA0494. For CRISPR-Cas9 editing:

  • Target guide RNAs to exons encoding these domains

  • Prioritize sites with high on-target and low off-target scores

  • Consider introducing precise mutations in the calcium-binding motifs rather than creating complete knockouts

• Functional Domain Conservation Analysis:

  Domain	Mouse Position	Human Position	Conservation	Functional Significance
  EF-hand 1	AA 180-215	AA 182-217	92%	Primary calcium binding
  EF-hand 2	AA 290-325	AA 292-327	89%	Secondary calcium binding
  Transmembrane	AA 70-92	AA 71-93	95%	Membrane localization
  C-terminal	AA 400-484	AA 402-486	83%	Protein interactions

• HDR Template Design Considerations:

  • For knock-in experiments, preserve critical calcium-binding residues in the DxDxDG motifs

  • Design homology arms that avoid disrupting domain boundaries

  • Consider tagging strategies that don't interfere with calcium binding (C-terminal tags preferred)

• Verification Strategies:

  • Design PCR primers spanning edited regions

  • Create sequencing strategies that verify precise edits

  • Plan functional readouts specific to calcium binding (calcium imaging, protein interaction studies)

• Rescue Experiments:

  • Prepare expression constructs of wild-type mouse KIAA0494 for rescue experiments

  • Create domain-specific mutants to test function of individual domains

  • Consider human KIAA0494 for cross-species rescue experiments

These considerations ensure that gene editing experiments produce interpretable phenotypes directly related to the calcium-binding functions of KIAA0494.

How can mouse KIAA0494 be utilized as a research tool in calcium signaling studies?

Mouse KIAA0494 can be leveraged as a sophisticated research tool in calcium signaling studies through several innovative approaches:

• Calcium Sensor Development: The EF-hand domains of KIAA0494 can be engineered as novel calcium biosensors by:

  • Fusing them with fluorescent proteins to create FRET-based sensors

  • Incorporating environmentally sensitive fluorophores near calcium-binding sites

  • Creating chimeric proteins with optimized calcium affinities for specific applications

• Cellular Calcium Buffering Modulation: Recombinant KIAA0494 can be used to:

  • Systematically alter cellular calcium buffering capacity when overexpressed

  • Create dominant-negative constructs to interfere with endogenous calcium buffering

  • Study the effects of altered calcium dynamics on downstream signaling pathways

• Protein Interaction Platform: Tagged versions of the protein serve as excellent baits for:

  • Identifying novel components of calcium signaling complexes

  • Creating protein interaction maps under varying calcium concentrations

  • Developing pull-down assays to monitor dynamic changes in calcium-dependent protein complexes

• Structure-Function Analysis Tool: The well-defined EF-hand domains make KIAA0494 an excellent model for:

  • Studying the structural basis of calcium binding specificity

  • Investigating conformational changes induced by calcium binding

  • Teaching advanced protein structure concepts in research education

• Evolutionary Comparison Studies: Mouse KIAA0494 compared with human and other species provides:

  • Insights into the evolution of calcium signaling mechanisms

  • Understanding of conserved versus species-specific calcium regulation

By implementing these research applications, investigators can gain deeper mechanistic insights into calcium signaling beyond the direct physiological roles of KIAA0494 itself.

What are the most promising research directions for understanding KIAA0494's role in pathophysiological conditions?

The investigation of KIAA0494's role in pathophysiological conditions presents several promising research directions:

• Neurological Disorders:

  • Calcium dysregulation is implicated in numerous neurological conditions

  • Investigate KIAA0494 expression and mutation patterns in neurodegenerative disease models

  • Focus on regions showing high KIAA0494 immunoreactivity in brain tissue using validated antibodies

  • Study how KIAA0494 modulates neuronal calcium homeostasis using recombinant protein approaches

• Cardiovascular Pathologies:

  • Examine KIAA0494's role in cardiomyocyte calcium handling

  • Investigate potential associations with arrhythmias or heart failure

  • Study KIAA0494 interactions with established cardiac calcium channels and pumps

  • Use recombinant protein to identify cardiac-specific interacting partners

• Cancer Biology:

  • Analyze KIAA0494 expression across cancer types using immunohistochemistry

  • Investigate whether KIAA0494 affects calcium-dependent cell proliferation or apoptosis

  • Study the impact of KIAA0494 manipulation on cancer cell migration and invasion

  • Examine whether KIAA0494 modulates therapeutic responses to calcium-targeting drugs

• Immune System Dysfunction:

  • Study KIAA0494's role in calcium-dependent immune cell activation

  • Investigate its function in store-operated calcium entry in lymphocytes

  • Examine whether KIAA0494 variants associate with autoimmune conditions

  • Develop targeted immunomodulatory approaches based on KIAA0494 function

• Developmental Disorders:

  • Create animal models with KIAA0494 modifications to study developmental phenotypes

  • Investigate calcium signaling during embryogenesis in these models

  • Examine potential associations with human developmental disorders

  • Use recombinant protein to identify developmental stage-specific interactions