Recombinant Rat Sushi repeat-containing protein SRPX (Srpx)

What is Recombinant Rat SRPX protein and what are its basic characteristics?

Recombinant Rat SRPX (Sushi repeat-containing protein X-linked) is a protein expressed and purified from expression systems such as HEK293 cells. The protein contains sushi repeat domains (also known as complement control protein or CCP modules) and is encoded by the SRPX gene (Gene ID: 64316) in rats. Its UniProt ID is Q63769, and the corresponding mRNA Refseq is NM_022524.1 with a protein Refseq of NP_071969.1 .

The recombinant protein is typically produced with tags such as His, Fc, or Avi tags to facilitate purification and detection. When properly stored and handled, recombinant SRPX protein remains stable for at least 6 months. For optimal stability, the protein should be stored at -20°C to -80°C, avoiding repeated freeze-thaw cycles .

How does SRPX expression vary across different tissue types?

Research has demonstrated that SRPX protein expression correlates with tumor grade in gliomas, with significantly higher expression observed in glioblastoma (Grade 4) compared to lower-grade gliomas (Grades 2-3) . This differential expression pattern makes SRPX a potential biomarker for distinguishing between different grades of gliomas and potentially for monitoring disease progression.

Immunohistochemical staining (IHC) analysis of tumor tissue samples has confirmed this correlation between SRPX expression and tumor grade, providing visual evidence of SRPX upregulation in higher-grade tumors .

What experimental methods are recommended for handling and storing recombinant SRPX protein?

For optimal handling and storage of recombinant SRPX protein, researchers should follow these methodological guidelines:

• Storage conditions: Store the lyophilized protein at -20°C to -80°C. After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles, which can degrade protein quality and functionality .

• Reconstitution: Reconstitute lyophilized SRPX protein in PBS buffer. For certain applications, it may be beneficial to include a carrier protein such as BSA (bovine serum albumin) to enhance stability. Typical reconstitution concentrations are around 100 μg/mL .

• Stability considerations: Even with proper storage, plan experiments within 6 months of receipt, as protein activity may diminish over extended storage periods .

• Purity assessment: Before use in critical experiments, verify protein purity via SDS-PAGE. High-quality recombinant SRPX should show ≥85% purity .

• Endotoxin testing: For cell culture applications, confirm that endotoxin levels are below acceptable thresholds (typically <1.0 EU per μg protein) to prevent experimental artifacts from contamination .

How does SRPX function as a biomarker in glioblastoma research?

SRPX has emerged as a promising biomarker for glioblastoma through several key mechanisms and experimental findings:

• Exclusive presence in tumor-derived extracellular vesicles (EVs): Proteomics analysis has revealed that SRPX is the only protein consistently found in EVs derived from glioblastoma cell lines that is simultaneously absent in EVs derived from human primary astrocytes (HPAs) . This unique expression pattern provides a clear discriminatory marker for distinguishing tumor-derived EVs from those of normal brain cells.

• Correlation with tumor grade: Both mRNA and protein expression levels of SRPX show significant association with glioma tumor grade. Immunohistochemical staining demonstrates progressively increasing SRPX expression from Grade 2 to Grade 4 (glioblastoma) tumors, making it valuable for histopathological grading .

• Association with treatment resistance: Notably, temozolomide (TMZ)-resistant tumor tissues exhibit highly positive SRPX staining compared to other tumor grades. Furthermore, glioblastoma cells exposed to TMZ display enhanced SRPX gene expression, suggesting SRPX may play a role in the development of treatment resistance .

• Non-invasive detection potential: As SRPX is present in extracellular vesicles, it offers the potential for non-invasive detection through liquid biopsies (blood samples), which could revolutionize glioblastoma diagnosis and monitoring without requiring invasive brain tissue sampling .

For researchers investigating SRPX as a biomarker, integrating multiple detection methods including proteomics, RT-qPCR, and immunohistochemistry provides the most comprehensive assessment of SRPX expression in experimental and clinical samples.

What role does SRPX play in glioblastoma cell growth and chemoresistance?

SRPX appears to play critical roles in both glioblastoma cell proliferation and response to chemotherapy:

• Cell viability regulation: Knockdown of SRPX gene expression via siRNA has been demonstrated to inhibit glioblastoma cell viability, suggesting SRPX is essential for maintaining tumor cell growth and survival .

• Chemoresistance mechanism: Experimental evidence indicates that SRPX expression increases when glioblastoma cells are exposed to temozolomide (TMZ), the standard-of-care chemotherapy for glioblastoma. This upregulation appears to be dose-dependent and time-dependent .

• TMZ sensitivity modulation: Tissue samples from TMZ-resistant tumors show significantly higher SRPX expression compared to treatment-responsive tumors, indicating SRPX may contribute to the development of chemoresistance .

The relationship between SRPX expression and TMZ sensitivity can be experimentally demonstrated using the following methodology:

• Culture glioblastoma cells in a 96-well plate at a concentration of 5×10⁴ cells per well

• After 24 hours, expose cells to increasing TMZ concentrations (10μM, 25μM, 50μM, 75μM, 100μM, 200μM, 300μM)

• Incubate for 72 hours, then harvest cells and isolate RNA for SRPX expression analysis

• Alternatively, expose cells to a fixed TMZ concentration (200μM) and analyze SRPX expression at various time points (1h, 6h, 12h, 18h, 24h, 48h, 72h)

These methodological approaches allow researchers to precisely characterize the temporal and dose-dependent relationships between TMZ exposure and SRPX expression.

How can researchers effectively design SRPX knockdown experiments to study its function?

Designing effective SRPX knockdown experiments requires careful consideration of several methodological aspects:

• siRNA design and validation: Design multiple siRNA sequences targeting different regions of the SRPX mRNA to ensure specificity and efficacy. Always include a scrambled siRNA control. Validate knockdown efficiency using RT-qPCR and Western blot to confirm both mRNA and protein reduction.

• Transfection optimization: For glioblastoma cell lines, optimize transfection conditions (reagent concentration, cell density, incubation time) to achieve maximum knockdown with minimal cellular toxicity. Lipid-based transfection reagents typically work well for adherent glioblastoma cells.

• Functional assays: After confirming SRPX knockdown, assess multiple cellular functions including:

  • Cell viability (using MTT or similar assays)

  • Colony formation capability

  • Migration and invasion potential

  • Response to TMZ treatment at various concentrations

• Rescue experiments: To confirm phenotype specificity, perform rescue experiments by re-expressing SRPX using an expression vector containing an SRPX coding sequence resistant to the siRNA.

• In vivo validation: For comprehensive understanding, validate in vitro findings using orthotopic glioblastoma xenograft models with SRPX knockdown cells, monitoring tumor growth, invasiveness, and response to TMZ treatment.

When analyzing results, researchers should pay particular attention to the temporal dynamics of knockdown effects, as compensation mechanisms may emerge over extended periods.

What are the optimal expression systems for producing recombinant rat SRPX protein?

The selection of an appropriate expression system for recombinant rat SRPX production depends on experimental requirements for protein folding, post-translational modifications, and downstream applications:

• Mammalian expression systems: HEK293 cells represent the gold standard for recombinant rat SRPX production when proper protein folding and post-translational modifications are critical . This system ensures:

  • Proper disulfide bond formation in the sushi domains

  • Appropriate glycosylation patterns

  • Native-like conformation

• Insect cell systems: Baculovirus-infected insect cells (Sf9, Sf21, High Five) offer a compromise between mammalian systems and bacterial expression, providing some post-translational modifications with higher yield.

• Bacterial systems: While E. coli systems yield the highest protein quantities, they often produce misfolded SRPX requiring refolding protocols. This system is suitable primarily for applications where post-translational modifications are not critical.

The expression construct design should consider:

• Inclusion of appropriate secretion signals for extracellular expression

• Strategic placement of purification tags (N-terminal vs. C-terminal)

• Codon optimization for the selected expression system

For most research applications requiring functional studies of SRPX, mammalian expression systems like HEK293 cells provide the most reliable source of properly folded and modified protein .

What purification strategies yield the highest purity recombinant SRPX protein?

Achieving high-purity recombinant SRPX requires a multi-step purification strategy tailored to the protein's properties and the expression system used:

• Affinity chromatography as the initial capture step:

  • For His-tagged SRPX: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co²⁺ resins

  • For Fc-tagged SRPX: Protein A or Protein G affinity chromatography

  • For Avi-tagged SRPX: Streptavidin affinity chromatography

• Secondary purification to remove contaminants and tag cleavage (if required):

  • Size exclusion chromatography (SEC) to separate monomeric SRPX from aggregates and lower molecular weight impurities

  • Ion exchange chromatography (IEX) based on SRPX's theoretical isoelectric point

• Quality control assessments:

  • SDS-PAGE to confirm ≥85% purity

  • Western blot to verify identity

  • Endotoxin testing using LAL method (<1.0 EU per μg)

  • Mass spectrometry to confirm molecular integrity

For applications requiring ultra-high purity (>95%), a three-step purification protocol combining affinity chromatography, IEX, and SEC is recommended. The final product should be formulated in PBS buffer and stored as single-use aliquots to prevent freeze-thaw degradation .

How can researchers effectively detect SRPX in tumor tissues and extracellular vesicles?

Detection of SRPX in tumor tissues and extracellular vesicles (EVs) requires different methodological approaches due to the distinct nature of these samples:

• Tumor tissue detection:

  • Immunohistochemistry (IHC): Offers visualization of SRPX expression patterns within the tissue context. Use formalin-fixed paraffin-embedded (FFPE) sections with optimized antigen retrieval methods. Scoring systems (0 to 3+) can quantify expression levels across different tumor grades .

  • Western blotting: Provides semi-quantitative analysis of SRPX protein levels in tissue lysates.

  • RT-qPCR: Measures SRPX mRNA expression levels, requiring careful normalization to appropriate housekeeping genes.

• Extracellular vesicle detection:

  • EV isolation: Use ultracentrifugation, size exclusion chromatography, or commercial isolation kits to purify EVs from cell culture medium or patient serum.

  • Mass spectrometry: Provides unbiased proteomics analysis to identify SRPX in EVs .

  • ELISA: Allows quantitative measurement of SRPX in purified EVs.

  • Nanoparticle tracking analysis: Confirms EV size distribution and concentration.

• Validation approaches:

  • Multiple antibody validation: Use antibodies recognizing different epitopes to confirm specificity.

  • Knockdown controls: Include SRPX-knockdown samples as negative controls.

  • Comparison with normal tissues: Always include appropriate normal tissue controls.

For comprehensive characterization, researchers should employ a multi-modal approach combining at least two independent detection methods to confirm SRPX presence and quantity in experimental samples.

How might SRPX serve as a therapeutic target for glioblastoma treatment?

SRPX presents several promising avenues as a therapeutic target for glioblastoma based on current research findings:

• Direct targeting strategies:

  • Small molecule inhibitors: Designing compounds that disrupt SRPX protein-protein interactions or inhibit its functional domains.

  • Monoclonal antibodies: Developing antibodies that bind to extracellular SRPX to block its function or trigger immune-mediated elimination of SRPX-expressing cells.

  • RNA interference therapeutics: Employing siRNA or antisense oligonucleotides to downregulate SRPX expression, which has already shown efficacy in reducing glioblastoma cell viability in pre-clinical models .

• Combination approaches with standard therapy:

  • SRPX knockdown sensitizes glioblastoma cells to temozolomide (TMZ), suggesting that combining SRPX inhibition with standard chemotherapy could overcome treatment resistance .

  • Targeting SRPX in conjunction with radiation therapy may enhance treatment efficacy by eliminating resistant cell populations.

• Diagnostic-therapeutic combinations (theranostics):

  • Using SRPX as both a diagnostic marker and therapeutic target could enable personalized treatment approaches.

  • Patients with high SRPX expression could be identified through liquid biopsies (detecting SRPX in extracellular vesicles) and then treated with SRPX-targeting therapies.

The development of SRPX-targeting therapeutics would benefit from further mechanistic studies to elucidate:

• The precise molecular pathways through which SRPX promotes tumor growth

• Structural analysis of SRPX to identify druggable binding pockets

• In vivo validation using orthotopic glioblastoma models with SRPX inhibition

What experimental designs are most appropriate for studying SRPX functions in glioblastoma models?

When investigating SRPX functions in glioblastoma, researchers should consider these experimental design approaches:

• In vitro experimental designs:

  • Genetic manipulation studies: Use CRISPR-Cas9 for complete SRPX knockout, inducible shRNA for temporal control of knockdown, or overexpression systems to study gain-of-function effects .

  • Functional assays: Assess cell proliferation, migration, invasion, and apoptosis under various conditions (normoxia vs. hypoxia, with vs. without TMZ treatment).

  • Mechanistic investigations: Employ co-immunoprecipitation, ChIP-seq, or RNA-seq to identify molecular interactions and downstream pathways affected by SRPX.

• Ex vivo approaches:

  • Patient-derived organoids: Generate 3D organoids from glioblastoma patient samples with varying SRPX expression levels to study drug responses in a more physiologically relevant context.

  • Slice cultures: Use brain slice cultures with implanted glioblastoma cells (with SRPX manipulation) to observe invasion in a structured environment.

• In vivo experimental designs:

  • Orthotopic xenograft models: Implant SRPX-manipulated glioblastoma cells into the brains of immunocompromised mice to study effects on tumor growth, invasion, and survival.

  • Genetically engineered mouse models: Develop transgenic mice with conditional SRPX expression in the brain to study its role in tumorigenesis and progression.

• Translational research approaches:

  • Patient sample correlation studies: Analyze SRPX expression in patient samples and correlate with clinical outcomes, using both tissue samples and liquid biopsies (EVs in blood) .

  • Quasi-experimental designs: When randomized controlled trials are not feasible, consider pre-post designs with non-equivalent control groups or interrupted time series approaches .

For all experimental designs, appropriate controls, adequate sample sizes, blinding of assessors, and rigorous statistical analysis are essential to ensure valid and reproducible results.

What future research directions might expand our understanding of SRPX in cancer biology?

Several promising research directions could significantly advance our understanding of SRPX in cancer biology:

• Mechanistic investigations:

  • Elucidate the signaling pathways and protein interactions through which SRPX promotes tumor growth and chemoresistance.

  • Determine if SRPX functions differently in the intracellular environment versus when secreted in extracellular vesicles.

  • Investigate how SRPX expression is regulated at the transcriptional and post-transcriptional levels in response to cellular stress and chemotherapy.

• Expanded cancer types exploration:

  • Examine SRPX expression and function across different cancer types beyond glioblastoma to determine if it represents a common oncogenic mechanism.

  • Compare SRPX roles in primary versus metastatic tumors to understand its potential contribution to cancer dissemination.

• Clinical translation opportunities:

  • Develop and validate SRPX-based liquid biopsy approaches for non-invasive cancer detection and monitoring.

  • Conduct prospective studies correlating SRPX expression with treatment responses and patient outcomes.

  • Explore SRPX as a stratification marker for clinical trials, particularly for TMZ-based therapies .

• Technological innovations:

  • Apply single-cell analysis techniques to understand heterogeneity of SRPX expression within tumors.

  • Develop advanced imaging approaches to visualize SRPX dynamics in living cells and tissues.

  • Create biosensors to monitor SRPX activity in real-time during tumor progression and treatment.

• Therapeutic development pathways:

  • Screen for small molecule inhibitors of SRPX using high-throughput approaches.

  • Investigate antibody-drug conjugates targeting SRPX-expressing cells.

  • Explore the potential of SRPX-targeting chimeric antigen receptor (CAR) T-cells for glioblastoma immunotherapy.