Recombinant Epinephelus akaara Calmodulin (calm)

What is the structural characterization of recombinant Epinephelus akaara Calmodulin?

Recombinant Epinephelus akaara Calmodulin (calm) is a calcium-binding protein consisting of 149 amino acids with a molecular weight of approximately 16.8 kDa. The protein structure includes EF-hand motifs that are responsible for calcium binding. According to the product information, the recombinant protein expresses the full mature protein sequence corresponding to amino acids 2-149 . The UniProt ID for this protein is Q7T3T2, and it shares significant sequence homology with calmodulins from other vertebrate species .

The protein typically achieves >85% purity when analyzed by SDS-PAGE and can be expressed in various systems including E. coli, yeast, baculovirus, or mammalian cells, with E. coli being the most common expression system for research purposes .

How should recombinant Epinephelus akaara Calmodulin be stored and handled for optimal stability?

For optimal stability, consider the following research-validated protocols:

Storage conditions:

• Lyophilized form: Store at -20°C for up to 12 months

• Reconstituted liquid form: Store at -20°C for up to 6 months

• Avoid repeated freeze-thaw cycles

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening

• Reconstitute 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

• Prepare working aliquots and store at 4°C for up to one week

These conditions have been optimized to maintain protein integrity and biological activity, which is particularly important for functional studies involving calcium-binding capability.

What expression systems are most effective for producing Epinephelus akaara Calmodulin?

While multiple expression systems can be used to produce recombinant Epinephelus akaara Calmodulin, E. coli remains the most widely used for research applications due to several advantages:

How does Epinephelus akaara Calmodulin compare to calmodulin from other species?

Calmodulin is a highly conserved protein across species. Comparative analysis shows:

This high conservation suggests that E. akaara Calmodulin likely functions similarly to other vertebrate calmodulins in calcium-binding and interaction with target proteins .

What are the recommended protocols for using recombinant Epinephelus akaara Calmodulin in calcium-binding assays?

For calcium-binding assays with recombinant Epinephelus akaara Calmodulin, researchers should consider the following methodological approach:

Fluorescence-based calcium binding assay:

• Prepare recombinant Calmodulin at 1-10 μM in calcium-free buffer (typically 20 mM HEPES, pH 7.4, 100 mM KCl)

• Add incremental amounts of CaCl₂ (0.1-10 mM)

• Measure intrinsic fluorescence changes at excitation 280 nm and emission 320-340 nm

• Plot fluorescence intensity against calcium concentration to determine binding parameters

Isothermal Titration Calorimetry (ITC) method:

• Prepare 20-50 μM Calmodulin in buffer (20 mM HEPES, pH 7.4, 100 mM KCl)

• Titrate with 1-10 mM CaCl₂

• Record heat changes to determine binding affinity (Kd), stoichiometry, and thermodynamic parameters

These methods provide complementary data on calcium-binding properties and can help determine if the recombinant protein maintains its native calcium-binding functionality .

How can recombinant Epinephelus akaara Calmodulin be used to study calcium signaling in muscle development of grouper fish?

Calcium signaling plays a crucial role in muscle development of grouper fish, particularly in relation to growth performance. Research has shown that calcium signaling pathways involving calmodulin are important contributors to the growth of hybrid groupers .

Experimental approach:

• Tissue culture system:

  • Isolate primary muscle cells from Epinephelus akaara

  • Treat cultures with recombinant Calmodulin at varying concentrations (0.1-10 μM)

  • Measure changes in calcium transients using fluorescent calcium indicators

• Gene expression analysis:

  • Examine the expression of calcium signaling genes in response to calmodulin treatment

  • Key genes to analyze include RyR1, RyR3, TnC, TnI1, TnI2, TnT1, and TnT2

• Functional studies:

  • Use calmodulin inhibitors (W-7 or trifluoperazine) as controls

  • Compare calcium signaling response with and without recombinant calmodulin

Research findings indicate that in hybrid grouper species, genes involved in calcium signaling showed differential expression patterns related to growth performance:

This data suggests that calcium signaling components, particularly troponins that interact with calmodulin, are upregulated in fast-growing hybrid groupers compared to parent species .

What procedures are recommended for studying Calmodulin's role in temperature adaptation mechanisms of Epinephelus akaara?

Epinephelus akaara exhibits physiological and histological responses to temperature variations that may involve calmodulin-dependent pathways. Research methods to investigate this relationship include:

In vivo temperature challenge experiments:

• Acclimate juvenile E. akaara to different temperatures (25°C, 28°C, 31°C, and 34°C)

• Collect tissue samples at 2, 7, and 42 days

• Perform Western blot analysis for calmodulin expression

• Correlate calmodulin levels with biochemical parameters (GOT, GPT, GLU, TP, TG, TCHO, LDH, and ALP)

Ex vivo tissue explant studies:

• Prepare gill and liver tissue explants from E. akaara

• Expose to temperature ranges (25-34°C)

• Measure calmodulin expression and activation of downstream targets

• Correlate with histological changes

Research has documented significant changes in biochemical parameters at higher temperatures (34°C), with mortality observed after 42 days exposure:

These temperature-dependent physiological changes likely involve calcium signaling pathways where calmodulin plays a critical role as temperature sensor and signal transducer .

How can site-directed mutagenesis of recombinant Epinephelus akaara Calmodulin be used to study calcium ion interactions?

Site-directed mutagenesis offers a powerful approach to understand structure-function relationships in calmodulin's calcium-binding properties:

Recommended methodological workflow:

• Design of mutations:

  • Target conserved calcium-binding residues in EF-hand motifs

  • Common mutations include D→A substitutions in calcium-coordinating aspartate residues

  • Design primers with appropriate restriction sites (e.g., BamHI and EcoRI for cloning)

• Mutagenesis protocol:

  • Transform into E. coli BL21(DE3) for expression

• Functional characterization:

  • Compare calcium-binding properties of wild-type and mutant proteins

  • Measure changes in thermal stability using differential scanning fluorimetry

  • Determine alterations in target protein interactions

This approach can help map the specific residues critical for calcium binding in E. akaara Calmodulin and identify any unique properties compared to other species.

What role does Calmodulin play in the reproductive development of Epinephelus akaara?

Epinephelus akaara exhibits complex reproductive biology as a protogynous hermaphrodite with bidirectional sex change capability. Calmodulin may be involved in these reproductive processes through calcium-dependent signaling pathways:

Experimental approaches to study this relationship:

• Gonadal expression analysis:

  • Compare calmodulin expression in different gonadal stages (immature, mature female, transitional, mature male)

  • Correlate with gonadosomatic index (GSI) changes

• In vitro gonad culture:

  • Treat gonad explants with recombinant calmodulin

  • Measure expression of reproductive hormones and sex differentiation genes

Research on cultured red spotted grouper has shown significant changes in GSI values during gonadal development:

These reproductive changes may involve calmodulin-mediated calcium signaling pathways that regulate vitellogenesis and gonadal development in response to environmental factors like temperature and photoperiod .

How can recombinant Epinephelus akaara Calmodulin be used in protein-protein interaction studies related to viral resistance?

Calmodulin may play a role in host-pathogen interactions, particularly with nervous necrosis virus (NNV) which affects grouper species. Methodological approaches include:

Co-immunoprecipitation (Co-IP) protocol:

• Prepare grouper tissue or cell lysates in non-denaturing conditions

• Add recombinant calmodulin with His or GST tag

• Capture protein complexes using appropriate affinity resin

• Identify interacting partners through mass spectrometry

Far-Western blot approach:

• Separate proteins from viral-infected tissues by SDS-PAGE

• Transfer to membrane and renature proteins

• Probe with biotinylated recombinant calmodulin

• Detect using streptavidin-HRP system

Studies with nervous necrosis virus capsid protein (NNVCP) identified 49 interacting proteins in grouper optic nerve tissues through immunoprecipitation. These proteins were involved in various cellular functions:

Similar approaches could be used to identify calmodulin-interacting proteins involved in viral resistance mechanisms .

What methodologies are recommended for investigating Calmodulin's role in muscle growth of hybrid grouper species?

Hybrid groupers often exhibit growth heterosis, with calcium signaling pathways potentially contributing to this enhanced growth. Methodological approaches to investigate calmodulin's role include:

Comparative expression analysis:

• Obtain muscle samples from hybrid grouper (e.g., E. fuscoguttatus × E. lanceolatus) and parent species

• Quantify calmodulin expression using qRT-PCR

• Compare with expression of downstream calcium signaling components

Functional validation in myocyte cultures:

• Isolate primary muscle cells from hybrid and parent groupers

• Treat with recombinant calmodulin or calmodulin inhibitors

• Measure myocyte differentiation, protein synthesis, and glycolysis rates

Research has shown that calcium signaling components are differentially expressed in hybrid groupers compared to parent species, with potential impacts on muscle growth:

These findings suggest that calmodulin-mediated calcium signaling, together with enhanced glycolysis, contributes to the superior growth of hybrid groupers compared to parent species .

What are the critical quality control parameters for recombinant Epinephelus akaara Calmodulin?

Ensuring proper quality control of recombinant E. akaara Calmodulin is essential for reliable research results. Key parameters include:

Purity assessment:

• SDS-PAGE analysis: Should show ≥85% purity with minimal contaminant bands

• Size exclusion chromatography: Monitor for aggregation or degradation products

Functional verification:

• Calcium-binding assay: Confirm binding of 4 calcium ions per molecule

• Target protein activation: Verify activation of a model calmodulin-dependent enzyme (e.g., phosphodiesterase)

Stability indicators:

• Thermal stability profile: Monitor unfolding temperature by differential scanning fluorimetry

• Storage stability: Test activity retention after storage at recommended conditions

Researchers should establish these quality control parameters before using the recombinant protein in complex experimental systems to ensure reproducible results .

How can researchers troubleshoot issues with recombinant Epinephelus akaara Calmodulin activity?

When experiencing reduced or inconsistent activity with recombinant E. akaara Calmodulin, consider the following troubleshooting approaches:

Systematic investigation of these factors can help identify and resolve issues affecting the performance of recombinant E. akaara Calmodulin in experimental settings .

What are the best practices for reconstituting lyophilized Epinephelus akaara Calmodulin to maintain functional integrity?

Proper reconstitution is critical for maintaining the functional integrity of lyophilized recombinant E. akaara Calmodulin:

Recommended reconstitution protocol:

• Allow the lyophilized protein to equilibrate to room temperature (15-30 minutes)

• Centrifuge the vial briefly to collect material at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Gently mix by swirling or inversion (avoid vigorous vortexing)

• Allow complete dissolution (5-15 minutes)

• For long-term storage, add glycerol to a final concentration of 5-50%

• Aliquot and store at -20°C or -80°C

Buffer considerations:

• For calcium-binding studies: 20 mM HEPES, pH 7.4, 100 mM KCl

• For target protein interaction studies: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl

• For calcium-free conditions: Include 1 mM EGTA

Following these practices will help ensure that the reconstituted protein maintains its native structure and calcium-binding properties .

What are emerging research areas for Epinephelus akaara Calmodulin in fisheries and aquaculture?

Several promising research directions are emerging for E. akaara Calmodulin with implications for fisheries and aquaculture:

Temperature adaptation in changing climate:

• Investigation of calmodulin's role in physiological responses to warming ocean temperatures

• Development of biomarkers for thermal stress based on calmodulin expression or modification

• Selection for temperature-resilient strains based on calmodulin variants

Reproductive control in aquaculture:

• Manipulation of calmodulin-dependent pathways to control sex change timing

• Development of non-hormonal approaches to reproduction management

• Understanding bidirectional sex change mechanisms involving calcium signaling

Disease resistance:

• Exploration of calmodulin's role in immune responses to pathogens

• Development of strategies to enhance viral resistance through calcium signaling modulation

• Identification of calmodulin interaction partners involved in host-pathogen responses

These emerging areas could lead to practical applications for improving E. akaara aquaculture practices and conservation strategies .

How might CRISPR-Cas9 gene editing techniques be applied to study Calmodulin function in Epinephelus akaara?

CRISPR-Cas9 gene editing offers powerful opportunities for studying calmodulin function in E. akaara:

Methodological approach:

• sgRNA design:

  • Target conserved regions of the calm gene

  • Design multiple sgRNAs to increase editing efficiency

  • Validate sgRNAs in cell culture before embryo injection

• Microinjection protocol:

  • Inject Cas9 protein:sgRNA complex into one-cell stage E. akaara embryos

  • Use concentrations of 250-500 ng/μL Cas9 and 50-100 ng/μL sgRNA

  • Include fluorescent markers for injection tracking

• Phenotypic analysis:

  • Assess calcium signaling using fluorescent indicators

  • Examine muscle development and growth patterns

  • Test response to temperature challenges

• Potential applications:

  • Generate calmodulin knockdown or knockout models

  • Create specific point mutations in calcium-binding domains

  • Develop reporter lines for calmodulin expression studies

This technology could provide unprecedented insights into calmodulin function in vivo and potentially lead to improved aquaculture strains with enhanced growth or stress resistance properties.

What computational approaches can be used to predict Calmodulin interactions in Epinephelus akaara?

Computational approaches offer cost-effective methods for predicting calmodulin interactions and functions:

Recommended computational strategies:

• Homology modeling:

  • Build 3D structure model of E. akaara Calmodulin based on crystal structures of homologous proteins

  • Validate model quality using PROCHECK, ERRAT, and VERIFY3D

  • Compare with other fish species calmodulin structures

• Molecular docking:

  • Identify potential binding partners from E. akaara transcriptome data

  • Perform docking simulations with calmodulin and target proteins

  • Score and rank interactions based on binding energy

• Molecular dynamics simulations:

  • Simulate calcium-binding dynamics at different temperatures

  • Model conformational changes upon target binding

  • Investigate effects of mutations on protein stability

• Machine learning approaches:

  • Train models to predict calmodulin-binding motifs in E. akaara proteins

  • Identify novel interaction partners based on sequence patterns

  • Predict functional effects of calmodulin variants