Recombinant Rat Vomeronasal type-1 receptor B13 (V1rb13)
Description
Ligand Specificity
V1rb13 detects sulfated steroids and pheromones, activating calcium-dependent signaling in VNO neurons . Studies show:
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Calcium Transients: Recombinant V1rb13 induces intracellular calcium flux in VNO sensory neurons, confirming ligand-receptor activation .
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Species-Specificity: Preferential activation by predator-derived cues (e.g., ferret stimuli) over conspecific signals .
Evolutionary Context
V1R receptors like V1rb13 have undergone gene loss in primates but retain functionality in rodents due to their reliance on VNO-mediated behaviors .
Expression and Purification
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Host System: Optimized for E. coli to ensure high yield (~1–2 mg/L culture) .
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Tagging: His-tagged for affinity chromatography, enabling >90% purity .
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Lyophilization: Stabilized in trehalose to prevent aggregation during storage .
Stability Data
| Condition | Recommendation |
|---|---|
| Short-term Storage | 4°C (up to 1 week) |
| Long-term Storage | -80°C in 50% glycerol |
| Freeze-Thaw Cycles | Avoid >3 cycles to prevent denaturation |
Behavioral Studies
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Predator Avoidance: V1rb13 activation triggers defensive behaviors in rats exposed to predator chemosignals .
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Pheromone Signaling: Used to map neural circuits involved in mating and aggression .
Biochemical Assays
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Calcium Imaging: Monitor real-time neuronal activation using fluorometric assays .
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Ligand Screening: Identify novel pheromones via receptor-ligand binding studies .
Limitations and Considerations
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate it in your order notes, and we will prepare the product accordingly.
Please note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please communicate this in advance. Additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
STRING: 10116.ENSRNOP00000047657
UniGene: Rn.10227
Q&A
What expression systems are optimal for producing functional recombinant V1rb13?
Table 1: Expression System Comparison
| System | Yield (mg/L) | Purity (%) | Functional Validation Method |
|---|---|---|---|
| E. coli | 5–10 | >90 | SPR with pheromone ligands |
| HEK293 | 1–2 | 85–90 | Calcium imaging |
How should researchers validate V1rb13’s ligand-binding specificity?
Use surface plasmon resonance (SPR) with immobilized V1rb13 on a Ni-NTA chip. Test putative ligands like 2-heptanone and 2,5-dimethylpyrazine at concentrations of 1 nM–10 µM. Include negative controls (e.g., solvent-only injections) to eliminate nonspecific binding artifacts. Data analysis requires fitting to a 1:1 Langmuir model, with acceptable χ² values <10 . Contradictions between SPR results and in vivo electrophysiology may arise due to missing co-receptors like Vmn2r1 .
What protocols ensure stable reconstitution of lyophilized V1rb13?
Reconstitute lyophilized protein in Tris/PBS buffer (pH 8.0) with 6% trehalose to prevent aggregation . For long-term storage, add 50% glycerol and aliquot to avoid >3 freeze-thaw cycles. Post-reconstitution, validate structural integrity via circular dichroism: expect α-helix peaks at 208 nm and 222 nm, with β-sheet signals <15% . Deviations indicate misfolding, requiring additional refolding steps with redox shuffling buffers.
How can researchers resolve discrepancies between in vitro binding assays and in vivo behavioral responses?
This paradox often stems from tissue-specific post-translational modifications absent in recombinant proteins. Implement two strategies:
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Glycosylation Profiling: Treat HEK293-expressed V1rb13 with PNGase F and compare binding kinetics to E. coli-derived protein .
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Co-receptor Reconstitution: Co-express V1rb13 with promiscuous Gα subunits (e.g., Gα15) in heterologous systems to amplify signaling cascades .
Table 2: Discrepancy Analysis Workflow
| Step | Method | Expected Outcome |
|---|---|---|
| 1 | SPR with native tissue extract | Identify missing binding partners |
| 2 | Phosphorylation site mapping | Detect activation-state markers |
| 3 | In situ hybridization | Verify endogenous expression zones |
What strategies enhance V1rb13’s stability for crystallography studies?
Crystallization requires monodisperse protein at ≥5 mg/mL. Use detergent screening (e.g., n-dodecyl-β-D-maltoside) to stabilize the transmembrane domain. Mutate flexible regions (residues 150–165) via alanine scanning, guided by homology modeling against solved GPCR structures . For cryo-EM, embed V1rb13 in nanodiscs with MSP1E3D1 scaffold protein to preserve native conformation .
How does V1rb13’s signaling pathway interact with downstream neuronal circuits?
Utilize in vivo optogenetic inhibition coupled with calcium imaging in transgenic V1rb13-Cre rats. Inject AAV-DIO-hM4Di into the vomeronasal organ and monitor c-Fos expression in the medial amygdala after ligand exposure. Quantify signal transduction latency (typically 200–500 ms) using GCaMP6f reporters . Unexpected delays (>1 s) suggest intermediate kinases like PKC-δ are involved, requiring phosphoproteomic validation.
What computational models predict V1rb13’s ligand repertoire?
Combine molecular docking (AutoDock Vina) and machine learning. Train a neural network on 200 known vomeronasal ligands using molecular descriptors (LogP, polar surface area). Validate top candidates (n ≥ 20) via SPR. Critical metrics:
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Receiver Operating Characteristic (ROC) AUC >0.85
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Matthews Correlation Coefficient (MCC) >0.4
False positives often derive from overfitting; address this by including decoy ligands in training sets .
Addressing Non-specific Binding in Affinity Purification
Problem: His-tag interactions with bacterial chaperones (e.g., GroEL). Solution: Incorporate 20 mM imidazole in lysis buffer and use tandem affinity tags (Strep-II + His). Validate via SDS-PAGE with Coomassie and anti-Strep Western blot .
Standardizing Functional Assays Across Laboratories
Adopt the IFRA (International Forum on Receptor Assays) guidelines:
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Use 10 nM fluorescent IP3 analog for Gq-coupled signaling quantification
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Normalize responses to maximum ATP-induced calcium release
Interpreting Evolutionary Divergence in V1rb13 Orthologs
Construct phylogenetic trees using maximum likelihood (RAxML) with 100 bootstraps. Key residues under positive selection (e.g., Phe209, Asp275) are identified via CodeML. Test these via site-directed mutagenesis and ligand-binding assays. Rat-specific substitutions (Gly115Arg) may alter ligand specificity compared to mouse orthologs .
