Recombinant Danio rerio 45 kDa calcium-binding protein (sdf4)

What is the basic structure and function of Danio rerio SDF4 protein?

SDF4 in Danio rerio is a 45 kDa calcium-binding protein that belongs to the CREC (Cab45/reticulocalbin/ERC45/calumenin) family. This protein consists of approximately 356 amino acids and primarily functions in regulating calcium-dependent activities in the endoplasmic reticulum lumen or post-ER compartments . The protein contains EF-hand motifs which are characteristic calcium-binding domains, enabling it to participate in calcium homeostasis and calcium-dependent signaling pathways within cells.

To study this protein's structure-function relationship, researchers typically use recombinant protein technology to express specific domains, such as the calcium-binding regions. For instance, expressing fragments similar to the Val184-Phe382 sequence (as done with rat SDF4) allows for targeted structural studies while maintaining functional calcium-binding properties .

How is SDF4 expression regulated during zebrafish development?

SDF4 expression patterns in zebrafish follow temporal and spatial regulation during development. While direct data from the search results doesn't specifically address SDF4 developmental regulation, analysis methods similar to those used in zebrafish pattern studies can be applied to track SDF4 expression.

Methodologically, researchers can monitor SDF4 expression from early embryogenesis through adult stages using techniques such as:

• Whole-mount in situ hybridization to visualize spatial expression patterns

• qRT-PCR for quantitative temporal expression analysis

• Transgenic reporter lines expressing fluorescent proteins under the SDF4 promoter

• Time-course studies starting from early developmental stages (21 dpf) through adult stages (66+ dpf)

This developmental timeline approach, similar to that used in pattern formation studies, allows for comprehensive understanding of when and where SDF4 becomes functionally important during zebrafish development.

What expression systems are most effective for producing recombinant zebrafish SDF4?

Based on comparative approaches used for related proteins, the most effective expression systems for recombinant zebrafish SDF4 include:

Prokaryotic expression systems:

• Escherichia coli represents the most widely used system for SDF4 expression, particularly for structural and functional studies . This system typically involves:

  • Cloning the SDF4 sequence into an expression vector containing a His-tag or other affinity tag

  • Transformation into BL21(DE3) or other expression-optimized E. coli strains

  • IPTG induction of protein expression

  • Lysis and affinity purification using the encoded tag

Eukaryotic expression systems:

• Yeast expression systems have demonstrated success with zebrafish proteins, yielding high purity (>90%) recombinant proteins . This approach is particularly valuable when post-translational modifications are essential for functional studies.

For optimization, researchers should consider:

• Expressing specific functional domains (such as AA 30-356) rather than the full protein when studying particular functions

• Including appropriate tags (His-tag being most common) to facilitate purification

• Evaluating protein solubility and folding in each system

What are the critical quality control parameters for recombinant zebrafish SDF4?

Quality control for recombinant zebrafish SDF4 requires comprehensive assessment of:

Purity verification:

• SDS-PAGE analysis to confirm size and purity (>95% purity recommended for functional studies)

• Western blotting to verify identity using anti-SDF4 or anti-tag antibodies

Functional validation:

• Calcium-binding assays to confirm proper folding and function

• Circular dichroism to assess secondary structure

• Thermal shift assays to evaluate protein stability

Activity assessment:

• Interaction studies with known binding partners such as calb2a, calb2b, s100b, which show high confidence interaction scores (0.806, 0.803, and 0.791 respectively)

• Functional assays specific to calcium-binding properties

For researchers developing standardized assays involving this protein, implementing statistical normalization approaches (as used in zebrafish behavioral studies) can minimize batch-to-batch variability and ensure experimental reproducibility .

How can recombinant SDF4 be used in zebrafish calcium signaling research?

Recombinant SDF4 serves as a powerful tool in calcium signaling research through multiple experimental approaches:

In vitro calcium flux analyses:

• Using purified recombinant SDF4 in calcium binding assays to determine binding kinetics and stoichiometry

• Competitive binding assays with other calcium-binding proteins to assess relative affinities

Ex vivo tissue studies:

• Application of labeled recombinant SDF4 to tissue sections to identify binding sites

• Calcium imaging in the presence of recombinant SDF4 to evaluate modulatory effects

Protein interaction studies:

• Pull-down assays using His-tagged SDF4 to identify novel interaction partners

• Competition assays with known partners including calb2a, calb2b, and s100b to map interaction domains

A comprehensive interaction network approach should evaluate SDF4's relationship with its predicted functional partners (illustrated in the table below):

Table 1: Top SDF4 interaction partners in zebrafish with confidence scores

What are effective strategies for studying SDF4 function in zebrafish development?

Studying SDF4 function in zebrafish development requires multiple complementary approaches:

Genetic manipulation:

• CRISPR/Cas9-mediated knockout or knockdown studies targeting SDF4

• Creation of transgenic zebrafish lines with tagged or modified SDF4

• Rescue experiments using recombinant SDF4 protein in knockout models

Temporal analysis:

• Developmental time-course studies from early stages (21 dpf) through adulthood (66+ dpf)

• Stage-specific manipulation of SDF4 expression using inducible systems

Behavioral phenotyping:

• Standardized behavioral assays similar to light/dark transition tests used in neuropsychopharmacology research

• Normalization approaches to account for inter-individual variability

• Specific phenotyping protocols with proper acclimation periods (e.g., 30-minute light acclimation)

Calcium imaging:

• Real-time calcium dynamics visualization in transgenic lines

• Correlation of calcium signaling patterns with developmental events

These approaches can be enhanced using topological data analysis and machine learning methods to quantify resulting developmental patterns, as demonstrated in zebrafish pattern formation studies .

How does zebrafish SDF4 interact with the endoplasmic reticulum stress response pathway?

Zebrafish SDF4, as a calcium-binding protein functioning in the ER and post-ER compartments, likely plays a significant role in the unfolded protein response (UPR) and ER stress pathways, though specific zebrafish data is limited in the search results. Based on homology with mammalian systems and known functional relationships:

ER stress response involvement:

• SDF4 may interact with key ER stress sensors including GRP78 and ATF6

• Pearson correlation analysis can be used to examine association between SDF4 expression and ER stress markers

• Calcium dysregulation during ER stress likely involves SDF4 modulation

Methodological approach to study this pathway:

• Generate recombinant zebrafish SDF4 protein with specific tags for interaction studies

• Perform co-immunoprecipitation with GRP78 and ATF6

• Conduct calcium imaging during induced ER stress with and without SDF4 overexpression

• Analyze expression correlation patterns using statistical methods such as logistic regression models

For researchers investigating SDF4 in stress response pathways, combining recombinant protein interaction studies with in vivo expression analysis would provide the most comprehensive mechanistic insights.

What are the post-translational modifications of zebrafish SDF4 and their functional significance?

Predicted post-translational modifications:

• N-linked glycosylation, potentially affecting protein folding and stability

• Phosphorylation sites that may regulate calcium-binding affinity

• Potential SUMOylation affecting protein localization

Experimental approaches to characterize PTMs:

• Expression of recombinant SDF4 in eukaryotic systems that preserve PTMs

• Mass spectrometry analysis of purified native and recombinant protein

• Site-directed mutagenesis of predicted modification sites followed by functional assays

• Comparative analysis with mammalian SDF4 modifications

To study glycosylation specifically, researchers should consider interaction studies with proteins like St6galnac2 (glycosyltransferase family), which shows an interaction confidence score of 0.669 with zebrafish SDF4 , suggesting potential glycosylation-related functional relationships.

How conserved is SDF4 structure and function between zebrafish and mammals?

SDF4 shows significant evolutionary conservation across vertebrates, making zebrafish an informative model for comparative studies:

Structural conservation:

Functional comparison:

• Both zebrafish and mammalian SDF4 regulate calcium-dependent activities in the ER and post-ER compartments

• Interaction networks show conservation of binding partners, particularly with calcium handling proteins

• Both likely participate in similar cellular processes including secretion and calcium homeostasis

Comparative experimental approach:

• Recombinant expression of both zebrafish and mammalian SDF4

• Structural analysis using X-ray crystallography or cryo-EM

• Cross-species rescue experiments in knockout models

• Side-by-side functional assays under identical conditions

This comparative approach can provide insights into both fundamental conservation and species-specific adaptations of calcium signaling systems.

How can recombinant zebrafish SDF4 studies contribute to understanding human disease mechanisms?

Zebrafish SDF4 research offers translational value for human disease studies through several mechanisms:

Disease modeling applications:

• Calcium signaling disorders, particularly those affecting the ER

• Neurodevelopmental conditions involving calcium dysregulation

• Potential role in stress response and inflammatory conditions

Translational research framework:

• Identify human disease-associated SDF4 variants

• Generate equivalent mutations in recombinant zebrafish SDF4

• Perform functional characterization of wild-type vs. mutant proteins

• Develop zebrafish models expressing the mutant forms

• Test therapeutic interventions targeting calcium homeostasis

Prognostic biomarker potential:

• Similar to findings in sepsis patients where SDF4 has been identified as a prognostic factor for mortality , zebrafish models could help elucidate mechanisms behind SDF4's role in disease progression

For researchers exploring SDF4 in disease contexts, combining recombinant protein studies with in vivo zebrafish models offers a powerful approach to validate findings and test interventions before advancing to mammalian systems.

What are the optimal buffer conditions for maintaining recombinant zebrafish SDF4 stability and function?

For researchers working with recombinant zebrafish SDF4, buffer optimization is critical for maintaining protein stability and functional integrity:

Recommended buffer components:

• Base buffer: 20-50 mM Tris-HCl or phosphate buffer (pH 7.4-7.6)

• Salt concentration: 150-300 mM NaCl to maintain solubility

• Calcium supplementation: 1-5 mM CaCl₂ to stabilize calcium-binding domains

• Reducing agents: 1-5 mM DTT or 2-10 mM β-mercaptoethanol to maintain disulfide bonds

• Glycerol: 5-10% to improve stability during freeze-thaw cycles

Storage considerations:

• Short-term (1-2 weeks): 4°C with protease inhibitors

• Long-term: Aliquot and store at -80°C, avoid repeated freeze-thaw cycles

• Flash freezing in liquid nitrogen with 10-15% glycerol recommended

Functional assay conditions:

• Calcium concentration must be carefully controlled and buffered

• pH stability should be maintained with adequate buffering capacity

• Consider protein concentration effects on oligomerization

While these recommendations are extrapolated from general protein biochemistry principles and approaches used for similar calcium-binding proteins, they provide a starting point for optimizing experimental conditions for zebrafish SDF4 research.

How can sex-specific differences impact SDF4 studies in zebrafish models?

When designing zebrafish studies involving SDF4, researchers should consider potential sex-specific differences that might influence experimental outcomes:

Sex-specific considerations:

• Zebrafish demonstrate sex-specific recombination rates , which may affect genetic manipulation strategies

• Hormonal influences may modulate calcium signaling pathways differently in male and female fish

• Expression levels of SDF4 and its interaction partners may vary between sexes

Recommended experimental approaches:

• Sex-matched experimental and control groups

• Separate analysis of data from male and female subjects

• Inclusion of sex as a variable in statistical analyses

• Investigation of hormone-dependent regulation of SDF4 expression

Statistical handling:

• Implement normalization strategies to account for sex-specific variability

• Consider larger sample sizes to detect potential sex-specific effects

• Apply appropriate statistical tests that can account for sex as a biological variable

These considerations become particularly important in translational research where findings in zebrafish models are intended to inform human disease mechanisms that may have sex-specific manifestations.