Recombinant Chicken SPRY domain-containing protein 7 (SPRYD7)

What is SPRYD7 and what cellular functions has it been associated with?

SPRYD7, also known as SPRY domain-containing protein 7, is a protein coding gene that contains the SPRY domain, which is believed to mediate protein-protein interactions. While SPRYD7 remains relatively understudied, recent research has identified its potential roles in cancer progression and metastasis, particularly in colorectal cancer (CRC). The protein has been associated with increased invasion and migration capabilities in cancer cells .

The human SPRYD7 protein consists of 157 amino acids and has several synonyms including CLLD6 and C13orf1 . In human studies, SPRYD7 overexpression has been linked to poor survival rates in CRC patients and with aggressive metastatic phenotypes . Specifically, functional proteomics characterization has demonstrated that SPRYD7 plays a key role in the invasion and migration of CRC cells and in liver homing and tumor growth.

In chicken models, SPRYD7 is being studied as a comparative model to understand conserved functions across species, though specific chicken SPRYD7 functions are still being elucidated through ongoing research.

What expression systems are most effective for producing recombinant chicken SPRYD7?

Several expression systems can be employed for the production of recombinant chicken SPRYD7, each with distinct advantages depending on your research objectives.

Mammalian expression systems such as COS-7 cells offer proper post-translational modifications and are suitable for functional studies. These cells can be cultured in DMEM containing 10% FBS, 1% non-essential amino acids, 1% L-glutamine, and antibiotics at 37°C in 5% CO₂ . Transfection can be performed using methods such as DEAE-dextran (600 μg/ml) with chloroquine (258 μg/ml) and appropriate plasmid DNA concentrations .

In vitro cell-free systems have also proven effective for producing recombinant proteins including SPRYD7, as indicated by commercial sources . These systems bypass cellular limitations and can efficiently produce proteins within hours.

For larger scale production, bacterial systems (E. coli) may be considered, though they lack post-translational modifications which may affect protein functionality. Baculovirus-insect cell systems represent a middle ground, providing some post-translational modifications with higher yield than mammalian systems.

The optimal choice depends on your specific research needs: functional studies typically benefit from mammalian systems, while structural studies might utilize bacterial systems for higher yields.

What are the recommended storage conditions and handling protocols for recombinant chicken SPRYD7?

Recombinant chicken SPRYD7, like many recombinant proteins, requires specific storage conditions to maintain stability and functionality. Based on established protocols for similar proteins, the following conditions are recommended:

Temperature: Store at -80°C for long-term preservation . This prevents protein degradation and maintains structural integrity.

Buffer composition: A suitable buffer for SPRYD7 preservation contains 50 mM Tris-HCl and 10 mM reduced Glutathione, with pH adjusted to 8.0 . This buffer composition helps maintain protein stability and prevents aggregation.

Aliquoting: It is critical to aliquot the protein upon receipt to avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of activity . Prepare single-use aliquots based on your experimental requirements.

Handling protocols:

• Thaw aliquots rapidly at 37°C and keep on ice when working with the protein

• Avoid vortexing, which can cause protein denaturation; instead, mix by gentle pipetting or inversion

• For experimental use, dilute in appropriate buffers immediately before use

• When designing experiments, include appropriate controls to account for buffer components that might affect your experimental system

Following these storage and handling recommendations will help ensure the maintenance of protein integrity and experimental reproducibility.

What purification methods yield the highest purity and activity for recombinant chicken SPRYD7?

Purification of recombinant chicken SPRYD7 requires a strategic approach to maximize both purity and biological activity. Based on established protein purification methodologies, the following multi-step process is recommended:

Initial capture: For GST-tagged recombinant chicken SPRYD7, glutathione affinity chromatography serves as an excellent first purification step . The protein can be captured using glutathione-agarose resin and eluted with buffer containing 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0.

Intermediate purification: Following affinity chromatography, ion exchange chromatography can be employed to remove contaminants with different charge properties. At physiological pH, the theoretical isoelectric point of SPRYD7 should be calculated to determine whether cation or anion exchange would be more appropriate.

Polishing step: Size exclusion chromatography (gel filtration) helps eliminate aggregates and further improves purity. A Superdex 75 or 200 column (depending on oligomerization state) running in a physiologically relevant buffer is recommended.

Quality control assessment should include:

• SDS-PAGE analysis with Coomassie staining to verify purity (target >95%)

• Western blot using anti-SPRYD7 antibodies to confirm identity

• Activity assays based on known SPRYD7 functions or binding partners

• Mass spectrometry to confirm protein integrity and molecular weight (expected molecular mass for GST-tagged human SPRYD7 is approximately 43.01 kDa)

Considering the limited research on chicken SPRYD7, optimization of these purification methods may be necessary through iterative testing and validation.

What experimental approaches are most effective for studying SPRYD7's role in disease models?

Investigating SPRYD7's role in disease models requires a comprehensive experimental approach that combines molecular, cellular, and in vivo techniques. Based on published methodologies for SPRYD7 and similar proteins, the following experimental strategies are recommended:

Genetic manipulation approaches:

• Stable overexpression: Establish genetically modified cell lines using selection markers such as G418 (1 mg/mL for selection, 0.6 mg/mL for maintenance) . This approach has successfully demonstrated SPRYD7's role in colorectal cancer progression.

• Transient silencing: Implement siRNA-mediated knockdown using 22 pmol siRNA with appropriate transfection reagents (e.g., JetPrime) . Validation of silencing should be performed via Western blot, qPCR, and semi-quantitative PCR 24 hours post-transfection.

• CRISPR/Cas9 gene editing: For complete knockout or endogenous tagging of SPRYD7 to study native protein interactions.

Functional assays:

• Migration and invasion assays to assess metastatic potential

• Proliferation and apoptosis assays to evaluate cell growth effects

• Angiogenesis assays, as SPRYD7 has been observed as an inductor of angiogenesis

Molecular interaction studies:

• Immunoprecipitation followed by mass spectrometry to identify protein interactors

• Proximity labeling techniques (BioID, APEX) to identify spatial protein interactions

• Yeast two-hybrid screening to identify direct binding partners

In vivo models:

• Orthotopic implantation models for cancer studies

• Intrasplenic injection of cells expressing SPRYD7 followed by assessment of liver homing capabilities

• Subcutaneous tumor growth models with external caliper measurements

Multi-omics integration:

• Combine proteomics (10-plex TMT quantitative proteins), transcriptomics, and metabolomics data to understand systemic effects of SPRYD7 manipulation

• Apply bioinformatics approaches to identify affected pathways and biological processes

This comprehensive approach enables researchers to elucidate SPRYD7's functional role across different biological contexts and disease models.

How can different protein tags affect the structure-function relationship of recombinant chicken SPRYD7?

The choice of protein tag for recombinant chicken SPRYD7 can substantially impact its structural integrity, functional activity, and experimental applicability. Understanding these effects is crucial for experimental design and interpretation.

Impact on protein structure:
N-terminal versus C-terminal tags can differentially affect protein folding and domain accessibility. For SPRYD7, which contains a SPRY domain important for protein-protein interactions, tag position is particularly critical. The SPRY domain must remain accessible for functional studies of binding partners.

Common tags and their effects:

• GST-tag (43.01 kDa for human SPRYD7): Provides excellent solubility and purification efficiency but its large size may interfere with certain functions . Consider GST removal via protease cleavage for functional studies.

• His-tag: Smaller size minimizes structural interference but may affect metal-binding properties if SPRYD7 naturally interacts with metal ions.

• Myc/DDK-tags: Useful for detection but may alter protein-protein interactions within cellular contexts.

Experimental considerations:

• For structural studies: Smaller tags or cleavable tags are preferred to minimize structural perturbations

• For protein-protein interaction studies: Compare results using different tag positions to identify potential artifacts

• For cellular localization studies: Verify that the tag doesn't mask localization signals

Validation approaches:

• Compare multiple tagged versions (N-terminal, C-terminal, different tag types)

• Include tag-removal experiments to confirm native function

• Perform circular dichroism or thermal shift assays to assess structural integrity

• Validate key findings with untagged protein when possible

When designing experiments with recombinant chicken SPRYD7, researchers should carefully consider tag effects and include appropriate controls to distinguish true biological effects from tag-induced artifacts.

What are the methodological challenges in analyzing SPRYD7 protein-protein interactions across species?

Investigating SPRYD7 protein-protein interactions across species presents several methodological challenges that require careful experimental design and validation. These challenges span from sample preparation to data interpretation:

Species-specific binding partner dynamics:
Chicken SPRYD7 likely has evolved distinct interaction networks compared to its human ortholog. When conducting cross-species experiments, researchers must consider evolutionary divergence in both SPRYD7 and its potential binding partners. Immunoprecipitation experiments may yield different results between species due to:

• Differential expression of binding partners

• Amino acid variations affecting binding interfaces

• Post-translational modification differences

Technological limitations:

• Antibody cross-reactivity: Commercial antibodies developed against human SPRYD7 may have reduced affinity for chicken SPRYD7, necessitating validation or development of species-specific antibodies

• Mass spectrometry databases: Chicken proteome databases may be less comprehensive than human databases, potentially limiting identification of novel binding partners

Experimental approaches to overcome challenges:

• Implement reciprocal co-immunoprecipitation using antibodies against both SPRYD7 and suspected binding partners

• Employ proximity labeling techniques (BioID, APEX) that work independently of antibody quality

• Utilize recombinant protein pull-down assays with purified proteins to confirm direct interactions

• Develop cross-linking mass spectrometry (XL-MS) protocols to capture transient interactions

Data analysis considerations:

• Apply stringent statistical filters to distinguish true interactors from background

• Implement comparative bioinformatics to identify conserved interaction motifs

• Validate key interactions through multiple complementary techniques

By addressing these methodological challenges systematically, researchers can generate reliable insights into the evolutionary conservation and divergence of SPRYD7 interaction networks.

What strategies can optimize SPRYD7 expression and purification from inclusion bodies?

When recombinant chicken SPRYD7 forms inclusion bodies during bacterial expression, a systematic approach to refolding and recovery is essential to obtain functionally active protein. The following strategies have proven effective for SPRY domain-containing proteins:

Optimized solubilization protocol:

• Harvest bacterial cells and lyse under native conditions to separate soluble and insoluble fractions

• Wash inclusion bodies thoroughly with detergent-containing buffers (e.g., 0.5% Triton X-100) to remove membrane proteins and debris

• Solubilize inclusion bodies using 8M urea or 6M guanidine hydrochloride in 50mM Tris-HCl, pH 8.0, with 1mM DTT or 5mM β-mercaptoethanol

Refolding optimization matrix:
Establish a multi-parameter refolding screen varying:

• Refolding method: Dilution, dialysis, or on-column refolding

• Buffer composition: Tris-HCl (pH 7.5-8.5) or phosphate buffer

• Additives: L-arginine (0.5-1M), glycerol (5-20%), sucrose (0.4-0.8M)

• Redox conditions: GSH/GSSG ratios (10:1, 5:1, 1:1) to facilitate disulfide bond formation

• Protein concentration: Lower concentrations (0.1-0.5 mg/mL) typically yield better results

The refolding success can be monitored by:

• Reduction in solution turbidity

• Increased fluorescence intensity (intrinsic tryptophan fluorescence)

• Circular dichroism to assess secondary structure recovery

• Functional assays specific to SPRYD7 activity

Purification after refolding:

• Remove aggregates via centrifugation (20,000g, 30 min) or filtration

• Apply the clarified refolded protein to appropriate affinity chromatography

• Consider additional purification steps (ion exchange, size exclusion) to isolate properly folded species

For SPRYD7 specifically, the presence of the SPRY domain suggests that protein-protein interaction functionality can serve as a useful metric for refolding success. Pull-down assays with known binding partners can validate the recovery of functional protein.

This systematic approach maximizes the recovery of correctly folded recombinant chicken SPRYD7 from bacterial inclusion bodies.

How can comparative proteomics be applied to elucidate SPRYD7 function in avian versus mammalian systems?

Comparative proteomics offers powerful insights into SPRYD7 function across species, revealing both conserved mechanisms and species-specific adaptations. A comprehensive comparative proteomics workflow between avian and mammalian systems should include the following methodological approaches:

Differential expression profiling:
Implement quantitative proteomics using isobaric labeling techniques such as TMT (Tandem Mass Tag) as demonstrated in SPRYD7 research :

• Extract proteins from equivalent tissues/cells from both species

• Perform tryptic digestion followed by isobaric labeling

• Fractionate peptides using high-pH reversed-phase chromatography

• Analyze by LC-MS/MS with synchronous precursor selection for enhanced identification

Data processing should include:

• Normalization to account for species differences

• Statistical analysis to identify significantly altered proteins

• Pathway analysis using tools compatible with both species

Interactome comparison:
To elucidate species-specific SPRYD7 interaction networks:

• Perform immunoprecipitation followed by mass spectrometry (IP-MS) in both species

• Implement proximity labeling approaches (BioID/APEX) in parallel systems

• Cross-reference interactors to identify:

  • Core conserved interactors (present in both species)

  • Species-specific interactors (unique to avian or mammalian systems)

Sample experimental design table:

Functional validation:

• Perform cross-species complementation assays (Can chicken SPRYD7 rescue phenotypes in mammalian cells?)

• Develop parallel cell-based assays to test functional conservation

• Utilize CRISPR screens in both systems to identify genetic interactions

This comprehensive comparative proteomics approach provides mechanistic insights into the evolutionary conservation and divergence of SPRYD7 function, potentially revealing species-specific adaptations and core biological roles.