SPARC Human, His

What is SPARC and what are its primary biological functions?

SPARC, also known as osteonectin or BM-40, is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. It functions as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture . The protein appears to regulate cell growth through interactions with the extracellular matrix and cytokines, binding calcium and copper, several types of collagen, albumin, thrombospondin, PDGF, and cell membranes .

The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. It contains two calcium binding sites: an acidic domain that binds 5 to 8 Ca²⁺ with low affinity and an EF-hand loop that binds a Ca²⁺ ion with high affinity . These calcium-binding properties are essential for its conformational changes and subsequent functions in matrix modulation.

Research methodologies to study SPARC functions include tissue-specific knockout models, cell culture systems with SPARC supplementation or depletion, and analysis of SPARC-dependent signaling pathways using phosphorylation-specific antibodies and pathway inhibitors.

What is the molecular structure of human SPARC protein?

Human SPARC protein consists of 303 amino acids with a calculated molecular weight of approximately 33.2 kDa, though it typically shows an observed molecular weight of about 38 kDa in SDS-PAGE due to post-translational modifications . The protein is organized into three distinct functional domains:

• An N-terminal acidic domain that binds multiple calcium ions with low affinity

• A follistatin-like domain containing disulfide-bonded structures

• An EF-hand domain with high-affinity calcium binding capability

The amino acid sequence of human SPARC protein is characterized by its richness in cysteine residues, which form essential disulfide bonds for maintaining its three-dimensional structure . The complete amino acid sequence includes:

MRAWIFFLLCLAGRALAAPQQEALPDETEVVEETVAEVTEVSVGANPVQVEVGEFDDGAEETEEEVAENPVCQNHHCKHGKVCELDENNPTPMCVCQDPTSCPAPIGEFEKVCSNDNKTFDSSCHFFATKCTLEGTKKGHKLHLDYIGPCKYPPCLDSELTEFPLRMRDWLKNVLVTLYERDEDNNLTEKQKLRVKKIHENEKRLEAGDHPVELLARDFEKNYNMYIFPVHWQFGQLDQHPIDGYLSHTELAPLAPMEHCTTRFFETCDLDNDKYIALDEWAGCFGIKQKDIDK

X-ray crystallography and NMR studies are recommended for detailed structural analysis of SPARC domains and their binding interfaces.

How is recombinant human SPARC protein with His tag produced and purified?

Recombinant human SPARC protein with histidine tag is typically produced using mammalian expression systems, particularly HEK 293 cells, to ensure proper folding and post-translational modifications essential for the protein's functional properties . The expression construct contains the full-length human SPARC sequence (Met1-Ile303) with a C-terminal histidine tag to facilitate purification .

The production process involves:

• Transfection of the expression vector containing the SPARC coding sequence into HEK 293 cells

• Culture of the transfected cells under controlled conditions for protein expression

• Harvesting of the cell culture medium containing the secreted recombinant protein

• Purification using immobilized metal affinity chromatography (IMAC) leveraging the His tag

• Additional purification steps such as size exclusion chromatography if needed

• Quality control testing including SDS-PAGE for purity assessment (>90% purity)

• Endotoxin testing using the LAL method to ensure levels below 1.0 EU/mg

• Lyophilization in a stabilizing buffer containing trehalose and mannitol

For researchers planning to express their own recombinant SPARC, optimization of calcium concentration in the culture medium is critical due to SPARC's calcium-binding properties affecting its folding and secretion.

What are the recommended storage and handling conditions for recombinant SPARC protein?

Proper storage and handling of recombinant human SPARC protein is crucial for maintaining its structural integrity and functional activity. Based on manufacturer recommendations, the following conditions should be observed:

• Lyophilized protein is generally stable for up to 12 months when stored at -20°C to -80°C

• For shipping, the lyophilized protein should be transported with ice packs

• Reconstituted protein solution can be stored at 4-8°C for 2-7 days

• For longer-term storage after reconstitution, the protein should be aliquoted to avoid freeze-thaw cycles and stored at temperatures below -20°C, where it remains stable for approximately 3 months

When handling the reconstituted protein:

• Maintain sterile conditions throughout to prevent microbial contamination

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation and aggregation

• Use appropriate low-protein-binding tubes for storage of dilute solutions

• Consider the addition of carrier proteins (such as BSA) for very dilute working solutions to prevent adhesion to container surfaces

Stability studies using analytical techniques such as size exclusion chromatography or dynamic light scattering are recommended to monitor protein quality over time.

What are the typical applications of recombinant human SPARC protein in research?

Recombinant human SPARC protein with His tag serves as a valuable tool in multiple research applications:

• ECM remodeling studies: Investigating SPARC's role as a matricellular chaperone in disassembly and degradation of ECM networks

• Cell-matrix interaction analysis: Examining how SPARC modulates cell adhesion, migration, and invasion through its interactions with extracellular matrix components

• Calcium signaling research: Studying the calcium-binding properties of SPARC and how they influence its conformational changes and functions

• Binding partner identification: Serving as bait in pull-down assays or surface plasmon resonance studies to identify novel SPARC-interacting proteins

• Antibody development and validation: Acting as an antigen for generating anti-SPARC antibodies for immunoassays and immunohistochemistry

• Functional assays: Supplementing cell cultures to assess SPARC's effects on cellular behavior including proliferation, differentiation, and apoptosis

• Structural studies: Providing material for crystallography, NMR, or cryo-EM studies to elucidate SPARC's three-dimensional structure and binding interfaces

Researchers should consider context dependency when designing experiments, as SPARC's effects may vary significantly depending on cell type and physiological context .

How does SPARC function as a matricellular chaperone in ECM remodeling?

The chaperone function of SPARC involves:

• Recognition and binding to specific ECM components, particularly collagens

• Modulation of collagen fibrillogenesis and fibril organization

• Regulation of matrix metalloproteinase (MMP) activity and localization

• Facilitation of ECM turnover by making matrix components more accessible to proteolytic enzymes

• Modulation of growth factor availability within the ECM

Experimental approaches to study this function include:

• Co-precipitation assays of SPARC with various ECM components under different calcium concentrations

• Real-time monitoring of ECM remodeling using fluorescently labeled matrix components in the presence of SPARC

• Atomic force microscopy to visualize structural changes in collagen fibrils mediated by SPARC

• Proteolytic susceptibility assays to assess how SPARC alters the degradation rate of ECM proteins

What methodologies are most effective for studying SPARC's calcium-binding properties?

SPARC contains two distinct calcium-binding domains with different affinities, making the study of its calcium-binding properties both complex and important for understanding its function. The following methodologies are particularly effective:

• Isothermal Titration Calorimetry (ITC): Provides direct measurement of binding thermodynamics and stoichiometry between SPARC and calcium ions, distinguishing between high and low-affinity binding sites.

• Circular Dichroism (CD) Spectroscopy: Detects secondary structural changes in SPARC upon calcium binding, offering insights into how calcium influences protein conformation.

• Fluorescence Spectroscopy: Using either intrinsic tryptophan fluorescence or extrinsic fluorescent probes to monitor calcium-induced structural alterations in real-time.

• Equilibrium Dialysis: Combined with atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) for quantitative determination of bound calcium.

• Site-Directed Mutagenesis: Systematic mutation of calcium-binding residues in both the acidic domain and EF-hand loop, followed by functional assays to determine the relative contribution of each binding site.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed structural analysis of calcium binding, particularly useful for examining the EF-hand domain.

• Differential Scanning Calorimetry (DSC): To assess how calcium binding affects SPARC's thermal stability.

Researchers should consider employing multiple complementary techniques to obtain a comprehensive understanding of SPARC's calcium-binding properties and their functional implications.

How can researchers assess SPARC's interactions with collagen and other ECM components?

Studying SPARC's interactions with collagen and other ECM components requires sophisticated methodological approaches that can capture both the specificity and dynamics of these interactions. Recommended approaches include:

• Solid-Phase Binding Assays:

  • Immobilize purified ECM components (collagens, thrombospondin, etc.) on microplates

  • Add varying concentrations of recombinant SPARC protein

  • Detect bound SPARC using anti-His antibodies or direct SPARC antibodies

  • Generate binding curves to calculate affinity constants

• Surface Plasmon Resonance (SPR):

  • Provides real-time kinetic data on association and dissociation rates

  • Can determine how calcium concentration influences binding parameters

  • Allows testing of multiple ECM components sequentially on the same chip

• Microscale Thermophoresis (MST):

  • Measures interactions in solution with minimal sample consumption

  • Particularly useful for studying calcium-dependent binding

• Proximity Ligation Assays:

  • For studying SPARC-ECM interactions in tissue sections or cultured cells

  • Provides spatial information about interaction sites

• Fluorescence Resonance Energy Transfer (FRET):

  • Labels SPARC and potential binding partners with compatible fluorophores

  • Monitors real-time interactions and conformational changes

• Electron Microscopy:

  • Visualizes direct effects of SPARC on collagen fibril organization

  • Immunogold labeling can locate SPARC binding sites on collagen fibrils

• Atomic Force Microscopy (AFM):

  • Measures binding forces between SPARC and ECM components at the single-molecule level

  • Can assess how these forces change with calcium concentration or pH

When designing these experiments, researchers should consider the physiological relevance of buffer conditions, calcium concentrations, and pH levels, as these factors significantly influence SPARC's binding behavior.

What experimental approaches can distinguish between the different functional domains of SPARC?

• Domain-specific recombinant proteins:

  • Express and purify individual domains of SPARC with His tags

  • Compare their binding properties and biological activities to those of the full-length protein

  • Use in competition assays with full-length SPARC to identify domain-specific interactions

• Site-directed mutagenesis:

  • Introduce point mutations in key residues of each domain

  • Create domain deletion mutants

  • Assess how these mutations affect specific functions (calcium binding, collagen interaction, cell adhesion modulation)

• Domain-specific antibodies:

  • Develop antibodies that recognize specific domains of SPARC

  • Use in blocking experiments to inhibit domain-specific functions

  • Apply in immunoprecipitation to identify domain-specific binding partners

• Proteolytic fragmentation:

  • Controlled proteolysis to generate domain-specific fragments

  • Analyze the function of these fragments in various assays

  • Compare activity profiles with recombinant domain proteins

• Biophysical characterization:

  • Differential scanning calorimetry to assess domain stability

  • Circular dichroism to monitor domain-specific structural changes

  • NMR spectroscopy for detailed structural analysis of individual domains

• Cross-linking studies:

  • Use domain-specific cross-linkers to identify proximity relationships

  • Map interaction surfaces between SPARC domains and binding partners

• Computational modeling:

  • Molecular dynamics simulations to predict domain behavior

  • Docking studies to identify potential interaction surfaces

Through systematic application of these approaches, researchers can develop a comprehensive understanding of how each domain contributes to SPARC's diverse functions in different physiological and pathological contexts.

How can researchers address the context-dependency of SPARC's activities in experimental design?

SPARC exhibits context-dependent and cell-type-dependent activities , presenting a significant challenge for researchers. To effectively address this complexity in experimental design:

• Multi-cellular model systems:

  • Compare SPARC effects across different cell types simultaneously

  • Utilize co-culture systems that recapitulate tissue-specific cellular interactions

  • Employ organoid models that better represent tissue architecture and cellular diversity

• Physiologically relevant conditions:

  • Maintain appropriate calcium concentrations that reflect tissue-specific environments

  • Consider the influence of pH, which varies across tissues and affects SPARC function

  • Include relevant ECM components that may modulate SPARC activity

• Temporal considerations:

  • Design time-course experiments to capture dynamic changes in SPARC activity

  • Use inducible expression systems to control the timing of SPARC introduction

  • Consider both acute and chronic effects of SPARC exposure

• Concentration gradients:

  • Test wide ranges of SPARC concentrations to identify potential threshold effects

  • Create spatial gradients of SPARC to mimic developmental or wound healing environments

• Combinatorial approaches:

  • Assess SPARC function in the presence of various growth factors and cytokines

  • Examine how SPARC interacts with other matricellular proteins and ECM components

  • Use factorial experimental designs to identify interaction effects

• In vivo validation:

  • Complement in vitro findings with tissue-specific conditional knockout models

  • Use tissue-specific promoters for SPARC expression in transgenic models

  • Consider humanized mouse models for studying human SPARC in specific contexts

• Systems biology approaches:

  • Employ multi-omics analyses to capture global cellular responses to SPARC

  • Develop computational models that integrate contextual variables

  • Use machine learning to identify patterns in context-dependent responses

Documenting and reporting all experimental conditions thoroughly is essential for reproducibility and meaningful comparison across studies investigating SPARC's complex biology.

What are the recommended protocols for reconstituting lyophilized SPARC protein?

Proper reconstitution of lyophilized SPARC protein is critical for maintaining its structural integrity and functional activity. The following protocol is recommended based on manufacturer guidelines:

• Preparation:

  • Allow the lyophilized protein vial to equilibrate to room temperature before opening

  • Briefly centrifuge the vial to collect all material at the bottom

  • Work in a sterile environment, preferably in a laminar flow hood

• Reconstitution procedure:

  • Add sterile water to the vial to prepare a stock solution of 0.5 mg/mL

  • Gently rotate or swirl the vial to dissolve the protein completely

  • Avoid vigorous shaking, vortexing, or pipetting, which can cause protein denaturation

  • Allow the solution to stand at room temperature for 5-10 minutes to ensure complete dissolution

• Concentration verification:

  • Measure the concentration by UV-Vis spectrophotometry

  • Use the theoretical extinction coefficient for SPARC protein for accurate determination

  • Consider measuring at both 280 nm (protein absorbance) and 320 nm (aggregate scatter)

• Post-reconstitution handling:

  • For immediate use, dilute in appropriate buffers (PBS with calcium is recommended)

  • For storage, prepare single-use aliquots to avoid freeze-thaw cycles

  • Use low-protein-binding tubes for dilute solutions

  • For concentrations below 50 μg/mL, consider adding carrier protein (e.g., 0.1% BSA)

• Storage after reconstitution:

  • Store at 4-8°C if using within 2-7 days

  • For longer storage, freeze aliquots at -20°C or below for up to 3 months

This reconstitution protocol ensures optimal protein quality for downstream applications while minimizing potential degradation or loss of activity.

How can the activity and integrity of SPARC protein be validated in experimental settings?

Validating the activity and integrity of SPARC protein before experimental use is essential for reliable results. A comprehensive validation strategy should include:

• Structural integrity assessment:

  • SDS-PAGE analysis under reducing and non-reducing conditions to confirm molecular weight and disulfide bond formation

  • Western blotting with domain-specific antibodies to verify full-length protein presence

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Thermal shift assays to assess protein stability and proper folding

• Functional validation:

  • Calcium-binding assays (ITC or fluorescence-based) to confirm proper calcium interaction

  • Collagen binding assays to verify ECM interaction capability

  • Cell adhesion modulation assays, as SPARC typically inhibits cell adhesion to certain substrates

  • Migration assays with appropriate cell types known to respond to SPARC

• Biochemical characterization:

  • Size exclusion chromatography to confirm monomeric state and absence of aggregates

  • Mass spectrometry to verify molecular weight and detect potential modifications

  • Limited proteolysis to assess proper folding (correctly folded proteins show characteristic digestion patterns)

  • Dynamic light scattering to evaluate homogeneity and hydrodynamic radius

• Comparative analysis:

  • Side-by-side comparison with previous lots of confirmed active SPARC protein

  • Benchmark against published activity data using standardized assays

  • Dose-response curves to establish expected potency ranges

• Endotoxin testing:

  • LAL assay to confirm endotoxin levels below 1.0 EU/mg for cell-based experiments

The specific validation tests performed should be tailored to the intended experimental applications, with more stringent validation required for complex functional studies compared to basic binding assays.

What analytical techniques are most effective for characterizing SPARC protein quality?

Comprehensive characterization of SPARC protein quality requires multiple complementary analytical techniques:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (target >90% purity)

  • Capillary electrophoresis for high-resolution separation

  • Reversed-phase HPLC for detecting hydrophobic variants

• Identity confirmation:

  • Western blotting with anti-SPARC and anti-His antibodies

  • Peptide mapping with mass spectrometric detection

  • N-terminal sequencing to confirm the correct start of the protein

  • MALDI-TOF mass spectrometry for molecular weight verification

• Higher-order structure analysis:

  • Circular dichroism spectroscopy for secondary structure composition

  • Fluorescence spectroscopy for tertiary structure assessment

  • Differential scanning calorimetry for thermal stability evaluation

  • Fourier-transform infrared spectroscopy (FTIR) for secondary structure analysis

• Aggregation and homogeneity evaluation:

  • Size exclusion chromatography to detect aggregates and oligomers

  • Dynamic light scattering for particle size distribution

  • Analytical ultracentrifugation for sedimentation profile analysis

  • Multi-angle light scattering for absolute molecular weight determination

• Post-translational modification analysis:

  • Glycosylation analysis using lectins or mass spectrometry

  • Phosphorylation detection using specific antibodies or mass spectrometry

  • Disulfide mapping to confirm correct disulfide bond formation

• Contaminant testing:

  • Host cell protein assays (ELISA-based)

  • DNA quantification assays for residual DNA

  • Endotoxin testing using LAL method (target <1.0 EU/mg)

The results from these analyses should be compiled into a comprehensive certificate of analysis that accompanies each batch of protein, facilitating experimental reproducibility and troubleshooting.

What are the most reliable methods for quantifying SPARC protein concentration?

Accurate quantification of SPARC protein concentration is essential for experimental reproducibility. Several methods offer complementary approaches:

• UV-Visible Spectrophotometry:

  • Primary method recommended for reconstituted SPARC protein

  • Uses calculated extinction coefficient based on amino acid composition

  • Measured at 280 nm with correction for scattering at 320 nm

  • Advantages: Rapid, non-destructive, requires small sample volume

  • Limitations: Interference from contaminants absorbing at similar wavelengths

• Colorimetric Protein Assays:

  • BCA (bicinchoninic acid) assay: Compatible with most buffers and detergents

  • Bradford assay: Faster but more sensitive to interfering substances

  • Lowry assay: Higher sensitivity but more complex protocol

  • Advantages: Generally more tolerant to buffer components than UV methods

  • Limitations: Destructive to the sample, requires standard curves, variable response depending on protein composition

• Amino Acid Analysis:

  • Gold standard for absolute protein quantification

  • Involves complete hydrolysis followed by HPLC analysis of amino acids

  • Advantages: Highest accuracy, independent of protein folding state

  • Limitations: Technically demanding, requires specialized equipment, destructive to sample

• ELISA-based Quantification:

  • Using validated anti-SPARC antibodies

  • Sandwich ELISA formats offer highest sensitivity and specificity

  • Advantages: Highly specific, works in complex mixtures, very sensitive

  • Limitations: Requires validated antibodies, potential epitope masking issues

• Quantitative Western Blotting:

  • Combines electrophoretic separation with immunodetection

  • Fluorescent secondary antibodies enable precise quantification

  • Advantages: Confirms correct molecular weight, can detect degradation

  • Limitations: More time-consuming, semi-quantitative unless carefully calibrated

For most research applications, a combination of UV spectrophotometry for routine measurements and a secondary method (such as BCA assay) for verification is recommended. Critical applications may warrant amino acid analysis or quantitative ELISA for the highest precision.

What cell-based assays are most informative for studying SPARC's biological activities?

Cell-based assays provide crucial insights into SPARC's biological functions in a physiologically relevant context. The following assays are particularly informative:

• Cell Adhesion Modulation Assays:

  • Seed cells on different ECM substrates (collagen, fibronectin) with/without SPARC

  • Quantify adherent cells after specific time points

  • Analyze focal adhesion formation using immunofluorescence

  • Rationale: SPARC typically inhibits focal adhesion formation and cell spreading

• Migration and Invasion Assays:

  • Transwell migration assays with SPARC gradients

  • Wound healing (scratch) assays with SPARC treatment

  • 3D invasion assays using collagen or Matrigel matrices

  • Time-lapse microscopy to track individual cell movements

  • Rationale: SPARC modulates cell motility in a context-dependent manner

• ECM Production and Remodeling Assays:

  • Quantify collagen and other ECM proteins secreted by cells with/without SPARC

  • Measure collagen fibril organization using second harmonic generation microscopy

  • Assess ECM stiffness using atomic force microscopy

  • Rationale: SPARC influences ECM production, organization, and mechanical properties

• MMP Activity Assays:

  • Zymography to measure MMP activation in the presence of SPARC

  • Real-time MMP activity assays using fluorogenic substrates

  • Immunolocalization of MMPs in SPARC-treated cultures

  • Rationale: SPARC modulates MMP activity and localization

• Growth Factor Signaling Modulation:

  • Phosphorylation assays for growth factor receptors with/without SPARC

  • Reporter assays for downstream signaling pathway activation

  • Growth factor binding/release studies in the presence of SPARC

  • Rationale: SPARC can sequester growth factors and modulate their signaling

• Calcium Signaling Assays:

  • Intracellular calcium imaging using fluorescent indicators

  • Measure calcium-dependent signaling pathway activation

  • Rationale: SPARC's calcium-binding properties may influence cellular calcium homeostasis

• Cell Survival and Apoptosis Assays:

  • Annexin V/PI staining for apoptosis detection

  • Caspase activity assays following SPARC treatment

  • Cell viability assays under stress conditions with/without SPARC

  • Rationale: SPARC can promote or inhibit apoptosis depending on context

When designing these assays, researchers should carefully consider cell type selection, SPARC concentration ranges, timing of assessments, and appropriate positive and negative controls to generate robust and physiologically relevant data.