Recombinant Mouse P2Y purinoceptor 13 (P2yr13)
Description
Recombinant Expression Systems and Production
Recombinant Mouse P2Y purinoceptor 13 can be produced through different expression systems, each offering specific advantages for research applications. The choice of expression system significantly impacts protein folding, post-translational modifications, and ultimate functionality of the recombinant protein.
coli Expression System
One common approach involves expressing the full-length mouse P2yr13 in Escherichia coli (E. coli) bacterial systems. This method typically yields high protein quantities and is relatively cost-effective . The recombinant protein expressed in E. coli systems is often provided as a lyophilized powder with purity levels greater than 90% as determined by SDS-PAGE analysis . This expression system is particularly useful when post-translational modifications are not critical for the intended application.
Mammalian Expression System (HEK293)
For applications requiring mammalian post-translational modifications, recombinant P2yr13 can be produced in Human Embryonic Kidney 293 (HEK293) cells . This expression system better preserves the native protein conformation and modification patterns, potentially yielding a protein with characteristics more closely matching the naturally occurring receptor. The purity of HEK293-expressed P2yr13 is typically ≥85% as determined by SDS-PAGE .
His-Tagged Format
The His-tagged format consists of the full-length mouse P2yr13 (1-377aa) fused to an N-terminal histidine tag . This format facilitates protein purification through metal affinity chromatography and can be detected using anti-His antibodies. The His tag is relatively small and typically has minimal impact on protein function, making it suitable for various experimental applications.
His(Fc)-Avi-Tagged Format
A more complex tagging system involves the addition of multiple tags: His, Fc, and Avi tags . This combination offers several advantages:
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The His tag facilitates purification
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The Fc fragment (derived from immunoglobulin) enhances protein stability and half-life
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The Avi tag allows for site-specific biotinylation, enabling highly specific capture in various assay formats
This multi-tagged format is particularly useful for complex experimental setups requiring highly specific protein manipulation or detection methods.
Biological Functions and Applications
P2Y purinoceptor 13 serves important biological functions as a G-protein coupled receptor activated by ADP . Recent experimental evidence suggests that P2Y13 plays a significant role in the regulation of lipoprotein metabolism . This finding highlights its potential importance in research related to lipid metabolism disorders and cardiovascular diseases.
Research Applications
Recombinant mouse P2yr13 proteins find applications in various research contexts:
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Receptor-Ligand Binding Studies: Investigating the interaction between P2Y13 and its ligands, particularly ADP.
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Signal Transduction Analysis: Examining the downstream effects of P2Y13 activation in various cell types.
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Antibody Production: Generating and validating antibodies against P2Y13 for immunodetection purposes.
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Lipoprotein Metabolism Research: Studying the role of P2Y13 in lipid transport and metabolism .
Detection Methods
For quantitative detection of native P2Y13 in biological samples, specialized ELISA kits are available with a detection range of 0.156 ng/ml to 10 ng/ml . These kits are optimized for detecting native P2Y13 in tissue homogenates, cell lysates, and other biological fluids, though they may have limitations in detecting recombinant proteins due to potential differences in tertiary structure .
Reconstitution Protocol
For lyophilized P2yr13 preparations, the following reconstitution protocol is recommended:
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Briefly centrifuge the vial before opening to bring contents to the bottom
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Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Add glycerol to a final concentration of 5-50% (recommended: 50%)
Buffer Composition
Recombinant P2yr13 is typically stored in:
These buffer compositions help maintain protein stability during storage and handling.
Purity Assessment
Purity is typically determined by SDS-PAGE analysis, with E. coli-expressed P2yr13 showing greater than 90% purity and HEK293-expressed P2yr13 showing ≥85% purity .
Endotoxin Testing
For preparations intended for cell culture or in vivo applications, endotoxin testing is critical. HEK293-expressed P2yr13 typically contains < 1.0 EU per μg of protein as determined by the Limulus Amebocyte Lysate (LAL) method .
Product Specs
Please note: We prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate it in your order remarks and we will fulfill your request to the best of our ability.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. Lyophilized form typically has a shelf life of 12 months at -20°C/-80°C.
Note: The complete sequence including tag sequence, target protein sequence and linker sequence could be provided upon request.
Target Background
- This study concludes that the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked acetylcholine release at motor nerve terminals are of the subtype P2Y13. This research provides new insights into the types of purinergic receptors involved in the fine-tuning of cholinergic transmission at mammalian neuromuscular junctions. PMID: 27058149
- The P2Y13 receptor demonstrates protection against atherosclerosis, primarily through its role in hepatobiliary reverse cholesterol transport. PMID: 25770145
- Activation of the P2Y13 receptor is crucial in mediating the high-fat-diet and palmitic acid-induced myenteric neuronal loss. PMID: 24510452
- Up-regulation of HDL-cholesterol metabolism via activation of the P2Y13R using agonists could promote reverse cholesterol transport and potentially inhibit atherosclerosis progression in mice. PMID: 24769858
- Findings indicate that the P2Y13 R plays a significant role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. PMID: 23629754
- The P2Y13 receptor regulates phosphate metabolism and FGF-23 secretion, influencing skeletal development. PMID: 24487286
- The regulation of P2Y13-R expression is studied, providing evidence that P2Y13-R is tightly regulated by constitutive ubiquitination in the ER, followed by proteasome degradation. PMID: 24030815
- The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading. PMID: 23362109
- Research demonstrates crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway. PMID: 22250198
- The P2Y13 receptor is implicated in the suppression of neuronal differentiation. P2Y13 receptor antagonists could be potential candidates for the treatment of neurodegenerative diseases. PMID: 22521313
- This study examines the role of the P2Y13 receptor in bone homeostasis both in vivo and in vitro. Deletion of the P2Y13 receptor leads to reduced trabecular bone in mice, affecting both osteoblasts and osteoclasts. PMID: 22108801
- Experiments assess the role of the purinergic receptor P2Y(13) in the regulation of lipoprotein metabolism, demonstrating that modulating its activity could benefit the treatment of dyslipidemia in individuals. PMID: 20817122
- Results highlight a previously unrecognized role of the purinergic P2Y13 receptor in inducing the Nrf2/HO-1 antioxidant response and providing protection against oxidative stress. PMID: 20447456
- This study shows that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output. Data indicate that P2Y13 activity is involved in macrophage-to-feces reverse cholesterol transport. PMID: 20830789
- The blockage of the P2ry13 protein receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in beta-cell survival. PMID: 19915796
- (p2y13 purinoceptor) PMID: 11961076
Q&A
What is P2Y purinoceptor 13 (P2yr13) and what are its primary functions?
P2Y purinoceptor 13 (P2Y13) is a G-protein coupled receptor that functions as a receptor for adenosine diphosphate (ADP) . It is coupled to G(i)-proteins and plays crucial roles in multiple biological systems. This receptor has been implicated in hematopoiesis and immune system regulation according to sequence homology studies . Recent research has revealed a particularly important role in neural stem cell (NSC) biology, where P2Y13 functions as a key regulator of the balance between quiescence and activation in adult neural stem cells . The receptor acts as a molecular switch that promotes NSC activation and lineage progression while simultaneously dampening self-renewal capacity .
What expression systems are used for producing recombinant mouse P2Y13 protein?
Several expression systems have been documented for producing recombinant mouse P2Y13:
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Mammalian Cell Expression: HEK-293 cells are commonly used for expressing recombinant mouse P2Y13 with a His tag (AA 1-337) . This system provides proper post-translational modifications essential for receptor functionality.
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Cell-free Protein Synthesis (CFPS): This system has been employed to produce mouse P2Y13 with a Strep Tag, yielding protein with >70-80% purity as determined by SDS PAGE, Western Blot and analytical SEC (HPLC) .
The expression system selection depends on the intended application, with mammalian systems generally preferred when conformational integrity and post-translational modifications are critical.
What are the key structural characteristics of mouse P2Y13 protein?
Mouse P2Y13 protein (P2ry13) has the following structural characteristics:
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Protein Structure: A G-protein coupled receptor with the characteristic seven transmembrane domain architecture
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N-terminal Sequence: MLGTINTTGM QGFNKSERCP RDTRMTQLLF PVLYTVVFLA GILLNTVALW VFVHIPSNST FIVYLKNTLV ADLIMALMLP FKILSDSHLA PWQLRGFVCT LSSVVFYETM YVGIMMLGLI AFDRFLKIIM PFRKTFVKKT AFAKTVSISV WSLMFFISLP NMILNKEATP SSVKKCASLK
The receptor contains specific regions involved in ligand binding and G-protein coupling that are essential for its signaling functions.
What experimental applications are recombinant P2Y13 proteins suited for?
Recombinant P2Y13 proteins can be utilized in several experimental applications:
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ELISA and Western Blot: Particularly those with His or Strep tags for detection using antibody-based methods
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Functional Studies: Though validation for all functional applications may not be complete, the recombinant proteins are expected to work for functional characterization
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Structural Studies: Purified recombinant P2Y13 can be used for structural analysis, especially when expressed in mammalian systems with >90% purity
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Binding Assays: To investigate interactions with ligands, antagonists, and other binding partners
The application should be matched to the expression system and purification tag selected.
How can P2Y13 expression be used to distinguish neural stem cell populations?
P2Y13 expression serves as a valuable marker to differentiate between quiescent neural stem cells (qNSCs) and activated neural stem cells (aNSCs) in the adult subependymal zone (SEZ) . The research by Paniagua-Herranz et al. demonstrates that:
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P2Y13 is specifically expressed in NSCs within the adult SEZ
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P2Y13 expression levels can be used to distinguish qNSCs from aNSCs
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The receptor shows preferential localization to the ventral wall of the SEZ, an area known to contain NSCs with higher neurogenic potential
For experimental applications, this differential expression pattern enables researchers to:
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Identify and isolate specific NSC subpopulations based on P2Y13 expression levels
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Track the transition from quiescence to activation in NSCs
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Potentially use P2Y13 as a marker to overcome challenges in distinguishing between mature astrocytes and qNSCs
What methodologies are recommended for investigating P2Y13's role in neural stem cell fate decisions?
Several methodological approaches have proven effective for studying P2Y13's influence on NSC fate decisions:
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Single-cell Tracking: Real-time tracking of NSCs and their progeny in the absence of added mitogens allows the construction of lineage progression trees and assessment of cell fate decisions at the single-cell level . This method can reveal:
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Balance between asymmetric and symmetric divisions
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Self-renewal versus differentiation outcomes
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Cell survival and death patterns
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Transition between quiescence and activation
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Genetic Manipulation: Overexpression or silencing of P2Y13 in NSCs can elucidate its functional role:
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Pharmacological Modulation: Using specific agonists or antagonists to modulate P2Y13 activity can help understand its signaling mechanisms .
What signaling pathways are affected by P2Y13 activity in neural stem cells?
P2Y13 receptor activity modulates several key signaling pathways in NSCs, as revealed by gene ontology (GO) term analysis following P2Y13 overexpression :
Additionally, P2Y13 overexpression upregulates:
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Growth factor receptors
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Galectin 3 (Lgals3) and galectin 3-binding protein (Lgals3bp), which enhance astrocyte plasticity
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Proteasome components (Psmb8, 9, 10), the preferred proteolytic pathway in activated NSCs
How does manipulation of P2Y13 expression affect neurogenesis and NSC behavior?
The effects of P2Y13 manipulation on NSC behavior have been well-documented and include:
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P2Y13 Overexpression:
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P2Y13 Blockade or Silencing:
These findings indicate that P2Y13 serves as a critical molecular switch in regulating the balance between quiescence and activation in adult NSCs, with significant implications for neurogenesis.
What are the technical considerations for purifying and storing recombinant P2Y13 protein?
When working with recombinant P2Y13 protein, several technical considerations should be addressed:
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Purification Methods:
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Storage Conditions:
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Buffer Considerations:
What experimental approaches can validate P2Y13 receptor functionality in neural stem cells?
To validate P2Y13 receptor functionality in NSCs, several experimental approaches can be employed:
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Calcium Imaging: Since P2Y13 is coupled to G(i)-proteins, inhibitory effects on cAMP production and subsequent calcium signaling can be measured
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Gene Expression Analysis: RNA sequencing or qPCR to assess changes in genes regulated by P2Y13 activity, particularly those involved in:
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Lineage Tracing: Using genetic fate mapping techniques to track NSC progeny following P2Y13 manipulation
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Pharmacological Validation: Using specific agonists and antagonists to modulate P2Y13 activity and observe effects on NSC behavior, providing evidence for receptor-specific effects
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In Vivo Electroporation: Local overexpression of P2Y13 in the SEZ can be achieved through in vivo electroporation, allowing for assessment of effects in the intact neurogenic niche
These approaches provide complementary evidence for P2Y13 functionality in regulating NSC behavior and neurogenesis.
What are emerging areas of investigation for P2Y13 in neurogenesis research?
Several promising research directions for P2Y13 in neurogenesis include:
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Translational Applications: Investigating whether P2Y13 can serve as a target for therapeutic interventions to enhance neurogenesis in neurodegenerative diseases
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Human Tissue Studies: Confirming P2Y13 expression patterns and functions in human neurogenic tissues, as the current research suggests it could potentially serve as a marker to distinguish qNSCs from mature astrocytes in humans
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Single-Cell Resolution Studies: Further exploring P2Y13's role in fate decisions at single-cell resolution, particularly in the context of neuronal subtype specification
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Interaction with Other Signaling Pathways: Investigating how P2Y13 signaling interacts with other established pathways that regulate NSC quiescence and activation
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Development of Specific Pharmacological Tools: Creating more specific agonists and antagonists for P2Y13 to overcome current limitations in studying this receptor due to the lack of highly specific modulators
Advancing these research areas will enhance our understanding of P2Y13's role in neurogenesis and potentially uncover new therapeutic targets for neurological disorders.
