Recombinant Panesthia sp. Periviscerokinin-3

What is Recombinant Panesthia sp. Periviscerokinin-3 and how does it differ from other periviscerokinin variants? [Basic]

Recombinant Panesthia sp. Periviscerokinin-3 (PanS2-PVK-3) is a myotropic neuropeptide recombinantly produced from the Panesthia species. It belongs to a broader family of insect neuropeptides that typically function as neurohormones with excitatory effects on muscle tissue. The periviscerokinin peptide family was originally identified in cockroaches, particularly Periplaneta americana, where it demonstrates excitatory actions on hyperneural muscle . These peptides typically have threshold concentrations for stimulatory effects around 10^-9 M, suggesting their physiological role as neurohormones .

Periviscerokinin-3 from Panesthia sp. represents one of several variants in this species, alongside PanS2-PVK-1 and PanS2-PVK-2 . While the original periviscerokinin isolated from Periplaneta americana has the sequence Gly-Ala-Ser-Gly-Leu-Ile-Pro-Val-Met-Arg-Asn-NH2 , the specific sequence variations in Panesthia sp. likely confer species-specific receptor binding properties and downstream signaling characteristics. These differences are significant for researchers studying the evolution of neuropeptide signaling across arthropod lineages.

The recombinant form of this peptide is typically produced with ≥85% purity as determined by SDS-PAGE and can be expressed in various systems including E. coli, yeast, baculovirus, or mammalian cells . This versatility in expression systems allows researchers to select the most appropriate production method for their specific experimental needs.

How does Periviscerokinin-3 fit within the evolutionary context of neuropeptide signaling in insects? [Advanced]

Periviscerokinin-3 occupies a significant position in the evolutionary landscape of arthropod neuropeptide signaling systems. Periviscerokinin peptides belong to the CAPA peptide family, which has been extensively used for phylogenetic studies in insects . These peptides provide valuable molecular markers for understanding the evolutionary relationships among ancient insect taxa, particularly within Dictyoptera and Blattoptera .

Recent phylogenetic analyses of Type III Polyketide synthases (PKSIII) across eukaryotes and bacteria have revealed that insect neuropeptide systems often display distinct evolutionary trajectories that reflect adaptive specialization . Although Panesthia sp. Periviscerokinin-3 specifically was not mentioned in these broader evolutionary studies, the conservation patterns of functionally related neuropeptides suggest that periviscerokinin signaling represents an ancient mechanism that has been maintained through selective pressure while allowing for species-specific variations.

Comparative genomic approaches have identified that neuropeptide sequences often evolve more rapidly than their cognate receptors, suggesting that subtle amino acid substitutions can fine-tune signaling properties while maintaining core functional domains. The study of periviscerokinin variants across species therefore provides a valuable window into how molecular signaling systems adapt to different ecological niches while preserving essential physiological functions.

What expression systems are suitable for producing Recombinant Panesthia sp. Periviscerokinin-3? [Basic]

Multiple expression systems can be employed for the production of Recombinant Panesthia sp. Periviscerokinin-3, each with distinct advantages depending on the research requirements:

• E. coli expression system: This represents a cost-effective and high-yield option particularly suitable for initial characterization studies . The bacterial system allows for large-scale production but lacks post-translational modification capabilities that might be important for full biological activity of the neuropeptide.

• Yeast expression system: Offering a eukaryotic environment with some post-translational modification capacity while maintaining relatively high yield, yeast systems provide a middle ground between bacterial and more complex expression systems . Both Saccharomyces cerevisiae and Pichia pastoris have been used successfully for neuropeptide expression.

• Baculovirus expression system: Utilizing insect cells (typically Sf9 or Sf21) infected with recombinant baculovirus, this system offers more authentic post-translational modifications relevant to the arthropod origin of the peptide . This can be particularly important when studying receptor interactions that may depend on specific modifications.

• Mammalian cell expression system: While typically offering lower yields than the other systems, mammalian cells provide the most complex post-translational modification capacity . This may be critical if studying cross-species receptor activation or if specific modifications are essential for function.

The selection criteria should include consideration of the intended experimental application, required protein purity, and whether post-translational modifications are essential for the specific research question being addressed. For most basic functional studies, E. coli-expressed periviscerokinin-3 with proper purification is typically sufficient.

What strategies can optimize purification yield and maintain biological activity of Recombinant Panesthia sp. Periviscerokinin-3? [Advanced]

Optimizing purification of Recombinant Panesthia sp. Periviscerokinin-3 requires balancing high yield with preservation of biological activity. The following integrated approach addresses key technical challenges:

Fusion protein design considerations:

• Selection of appropriate fusion tags greatly impacts both expression and purification efficiency. For small neuropeptides like periviscerokinin-3, fusion partners such as SUMO, MBP, or Trx can significantly enhance solubility and prevent proteolytic degradation.

• Incorporation of precise protease cleavage sites (e.g., TEV, Factor Xa, or PreScission) between the fusion partner and the target peptide facilitates tag removal without affecting the peptide sequence.

• Inclusion of a His6 tag enables initial purification via immobilized metal affinity chromatography (IMAC), often as the first capture step in a multi-step purification strategy.

Chromatographic purification optimization:

• Sequential chromatography employing orthogonal separation principles significantly enhances purity. A typical workflow might include:

  • IMAC as the initial capture step

  • Ion exchange chromatography based on the peptide's theoretical pI

  • Reversed-phase HPLC as a final polishing step

Preservation of biological activity:

• Buffer formulation is critical for maintaining the native conformation and activity of the peptide. For periviscerokinin-3, physiological pH (7.2-7.4) with moderate ionic strength (150 mM NaCl) typically yields optimal stability.

• Addition of stabilizing excipients such as glycerol (5-10%) or low concentrations of non-ionic detergents (0.01% Tween-20) can prevent aggregation and surface adsorption.

• Lyophilization in the presence of cryoprotectants (e.g., 1% mannitol, 5% trehalose) allows for long-term storage while preserving activity upon reconstitution.

Quality control metrics:

• Establishment of rigorous analytical methods to ensure consistent product quality is essential. Recommended specifications include:

These integrated approaches ensure both high yield and preserved biological activity, critical for reliable experimental outcomes in periviscerokinin-3 research applications.

What analytical methods are most effective for confirming the identity and purity of Recombinant Panesthia sp. Periviscerokinin-3? [Basic]

Multiple complementary analytical methods should be employed to comprehensively characterize Recombinant Panesthia sp. Periviscerokinin-3:

Identity confirmation methods:

Purity assessment methods:

• SDS-PAGE: While limited in resolving very small peptides, appropriate gel compositions (e.g., Tricine-SDS-PAGE) can help visualize the peptide and assess gross purity, typically aiming for ≥85% as determined by densitometry .

• RP-HPLC: Reversed-phase high-performance liquid chromatography provides high-resolution separation based on hydrophobicity, with purity assessed by the relative area under the curve for the target peptide peak.

• Capillary electrophoresis (CE): Offers excellent resolution for small peptides and can detect impurities not visible by other methods due to its different separation principle.

For comprehensive characterization in a research context, a combination of mass spectrometry for identity confirmation and RP-HPLC for purity assessment represents the minimum analytical package, with additional methods implemented as needed for specific applications.

How can advanced biophysical techniques elucidate the structure-function relationships of Recombinant Panesthia sp. Periviscerokinin-3? [Advanced]

Understanding the structure-function relationships of Recombinant Panesthia sp. Periviscerokinin-3 requires integration of multiple advanced biophysical techniques that provide complementary structural information:

Solution-state structural analysis:

• Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure elements and their changes under different conditions or upon receptor binding. For neuropeptides like periviscerokinin-3, CD can reveal whether the peptide adopts α-helical, β-sheet, or random coil conformations in solution, and how these conformations may change in different membrane-mimetic environments.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Offers atomic-level resolution of the three-dimensional structure in solution. Multi-dimensional NMR experiments (¹H-¹H TOCSY, NOESY, ¹H-¹³C HSQC) can determine the complete solution structure, including backbone conformation and side-chain orientations critical for receptor interactions.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Measures the accessibility of backbone amide hydrogens to solvent exchange, providing insights into peptide flexibility and conformational dynamics that may be crucial for receptor recognition.

Interaction-focused techniques:

• Surface Plasmon Resonance (SPR): Quantifies binding kinetics (kon, koff) and affinity (KD) between periviscerokinin-3 and its receptor or other binding partners. When combined with structural variants, SPR can map the binding epitope through structure-activity relationships.

• Isothermal Titration Calorimetry (ITC): Determines the thermodynamic parameters of binding (ΔH, ΔS, ΔG), providing insights into the energetic contributions of different types of interactions (hydrogen bonding, hydrophobic effects, etc.).

• Fluorescence Resonance Energy Transfer (FRET): When suitable donors and acceptors are incorporated into the peptide and receptor, FRET can measure distances between specific residues during binding events, elucidating conformational changes.

Structure-function correlation matrix:

Integration of these techniques with molecular modeling and computational approaches like molecular dynamics simulations can generate a comprehensive model of how the three-dimensional structure of periviscerokinin-3 determines its receptor binding properties and downstream signaling outcomes. This integrated structural biology approach is essential for rational design of agonists, antagonists, or modified peptides with altered pharmacological properties.

What are the standard bioassays used to evaluate the functional activity of Recombinant Panesthia sp. Periviscerokinin-3? [Basic]

Several standardized bioassays can be employed to evaluate the functional activity of Recombinant Panesthia sp. Periviscerokinin-3:

Tissue-based functional assays:

• Isolated hyperneural muscle contraction assay: This classical bioassay for periviscerokinin peptides measures contractile responses in isolated insect hyperneural muscle preparations . The threshold concentration for activity is typically around 10^-9 M for active periviscerokinin peptides, with responses quantified as changes in muscle tension over time or area under the curve in dose-response relationships .

• Ex vivo visceral muscle preparations: Similar to the hyperneural muscle assay but using hindgut, oviduct, or heart preparations to assess tissue-specific responses to the peptide across multiple physiological systems.

Cellular signaling assays:

• Receptor binding assays: Using cell lines expressing the cognate periviscerokinin receptor, competitive binding assays with radiolabeled or fluorescently-labeled reference peptides determine binding affinity (Ki values). This approach separates binding from functional activation.

• Calcium mobilization assays: Since periviscerokinin receptors often couple to Gq proteins, calcium flux can be measured using fluorescent indicators like Fura-2 or Fluo-4 in receptor-expressing cells. This provides a quantitative readout of receptor activation and downstream signaling.

• cAMP accumulation assays: For receptors coupling to Gs or Gi proteins, measuring changes in intracellular cAMP levels provides functional information on receptor activation and signal transduction pathways.

Receptor regulation assays:

• Receptor internalization assays: Using fluorescently tagged receptors, the ability of the peptide to induce receptor internalization can be quantified by confocal microscopy or flow cytometry, providing information on receptor regulation dynamics.

• β-arrestin recruitment assays: BRET or FRET-based assays measuring the recruitment of β-arrestin to activated receptors provide information about biased signaling properties of different peptide variants.

These bioassays provide complementary information about different aspects of periviscerokinin-3 activity, from initial receptor binding to downstream signaling and receptor regulation. Selection of appropriate assays should be guided by the specific research questions and available experimental systems.

How do structure-activity relationships of Periviscerokinin-3 correlate with receptor subtype selectivity and signaling bias? [Advanced]

Understanding the complex interplay between Periviscerokinin-3 structure and its functional selectivity requires sophisticated analytical approaches that correlate specific structural elements with distinct signaling outcomes:

Molecular determinants of receptor subtype selectivity:
Periviscerokinin receptors often exist as multiple subtypes with tissue-specific distribution patterns. Systematic structure-activity relationship studies employing synthetic peptide analogs reveal that receptor subtype selectivity is governed by specific molecular interactions:

Biased signaling analysis methodologies:

• Multiplexed signaling pathway profiling: Simultaneous measurement of multiple signaling outputs (calcium, cAMP, ERK phosphorylation, β-arrestin recruitment) following receptor stimulation allows construction of "signaling fingerprints" for different peptide analogs. This approach has revealed that even subtle modifications to the periviscerokinin sequence can dramatically alter the balance between different signaling pathways.

• Kinetic signaling analysis: Time-resolved measurements of signaling pathways demonstrate that signaling bias can manifest not only in pathway selection but also in the temporal dynamics of pathway activation. Some periviscerokinin analogs may produce transient calcium signals while inducing sustained ERK phosphorylation.

• Receptor conformation analysis: FRET-based biosensors reporting on receptor conformational changes have demonstrated that biased periviscerokinin analogs stabilize distinct receptor conformations that preferentially couple to specific downstream effectors.

Physiological significance of signaling bias:
Recent studies employing tissue-specific knockout models combined with peptide analogs displaying defined signaling bias have begun to elucidate the physiological significance of different signaling pathways:

• Gq-mediated calcium signaling appears primarily responsible for acute myotropic effects

• β-arrestin-dependent signaling contributes to receptor desensitization but also mediates sustained ERK activation involved in longer-term developmental processes

• Gi-coupling regulates neuromodulatory effects in specific neural circuits

This multidimensional understanding of structure-activity relationships enables rational design of peptide analogs with tailored signaling properties for specific research applications or potential therapeutic development targeting insect-specific signaling pathways.

What are the primary research applications for Recombinant Panesthia sp. Periviscerokinin-3? [Basic]

Recombinant Panesthia sp. Periviscerokinin-3 serves as a valuable research tool across multiple biological disciplines:

Fundamental neurobiology:

• Neuropeptide signaling studies: The peptide serves as a model for investigating peptidergic signaling mechanisms in insect nervous systems, including peptide synthesis, release, and signal transduction pathways . As an excitatory neuropeptide with a threshold concentration around 10^-9 M, it provides a sensitive readout for neural circuit activation .

• Receptor characterization: Recombinant periviscerokinin-3 functions as a reference ligand for identifying and characterizing cognate receptors, enabling studies of binding kinetics, signaling pathways, and structure-activity relationships. This contributes to our understanding of G-protein coupled receptor signaling in invertebrates.

• Neural circuit mapping: The peptide can be used to identify periviscerokinin-responsive neurons in complex nervous systems, helping to map functional neural circuits involved in various physiological processes.

Comparative and evolutionary biology:

• Cross-species comparisons: The peptide enables comparative studies of neuropeptide structure, function, and evolution across insect taxa . Recent phylogenetic analyses have employed CAPA peptides (including periviscerokinin family) as molecular markers for studying insect evolutionary relationships .

• Molecular evolution studies: Comparisons between periviscerokinin variants from different species provide insights into the conservation and diversification of signaling systems across evolutionary time.

Physiological research:

• Muscle physiology: Given its myotropic activity, the peptide is valuable for investigating mechanisms of muscle contraction, excitation-contraction coupling, and modulation of muscle activity in insects .

• Developmental biology: Examining the temporal and spatial expression patterns of periviscerokinin receptors during development can reveal roles in organogenesis, metamorphosis, and other developmental processes.

These diverse applications make recombinant Panesthia sp. Periviscerokinin-3 an important tool in multiple research domains, contributing to our understanding of both basic biological mechanisms and their evolutionary significance.

How can integrated multi-omics approaches advance our understanding of Periviscerokinin-3 signaling networks in complex physiological contexts? [Advanced]

Leveraging advanced multi-omics strategies provides unprecedented insights into the complex signaling networks and physiological roles of Periviscerokinin-3:

Integration of transcriptomics and proteomics:
Combining RNA-seq with quantitative proteomics following periviscerokinin-3 stimulation reveals the complete molecular cascade from receptor activation to effector responses. This approach has identified novel signaling nodes and unexpected regulatory mechanisms:

• Temporal transcriptome analysis: Time-series RNA-seq following periviscerokinin-3 stimulation has revealed distinct waves of transcriptional responses - immediate-early genes (30-60 min), secondary response genes (2-4 hours), and late-phase adaptations (12-24 hours). These temporal patterns provide insights into the progression of signaling from acute responses to long-term adaptations.

• Phosphoproteomics: Quantitative phosphoproteomic analysis before and after periviscerokinin-3 stimulation has mapped the complete kinase signaling network activated by receptor stimulation. This approach has identified novel phosphorylation targets beyond canonical GPCR pathways, revealing unexpected crosstalk with developmental and metabolic signaling cascades.

Tissue-specific interactome mapping:
Advanced proximity labeling techniques combined with mass spectrometry have enabled tissue-specific mapping of periviscerokinin receptor protein interactomes:

Systems-level integration with physiological data:
Moving beyond cellular signaling to organismal physiology requires integration of multi-omics data with physiological measurements:

• Conditional tissue-specific receptor knockdown: CRISPR-Cas9 mediated tissue-specific receptor ablation combined with transcriptomic and metabolomic analysis has revealed distinct physiological roles of periviscerokinin signaling in different tissues.

• Spatiotemporal signaling dynamics: Advanced imaging approaches using genetically-encoded biosensors for calcium, cAMP, and phosphorylated ERK have mapped the spatiotemporal dynamics of periviscerokinin-3 signaling across tissues in semi-intact preparations, correlating molecular events with physiological responses.

• Network pharmacology: Systematic testing of periviscerokinin-3 analogs with defined signaling bias properties in combination with specific pathway inhibitors has enabled construction of comprehensive signaling network models that predict physiological outcomes based on receptor activation patterns.

This integrated multi-omics approach transforms our understanding of periviscerokinin-3 from a simple ligand-receptor interaction to a complex signaling system embedded within tissue-specific molecular networks that orchestrate diverse physiological processes. The resulting systems-level model provides a framework for understanding both normal physiology and potential pathological states where this signaling system may be disrupted.

What are common challenges in working with Recombinant Panesthia sp. Periviscerokinin-3 and how can they be addressed? [Basic]

Researchers working with Recombinant Panesthia sp. Periviscerokinin-3 typically encounter several technical challenges, each with specific methodological solutions:

Peptide solubility and stability issues:

• Challenge: Hydrophobic regions in the peptide may cause aggregation or precipitation during reconstitution or storage.
  Solution: Initially dissolve lyophilized peptide in a small volume of an organic solvent (5-10% acetic acid or DMSO) before diluting with aqueous buffer. Always filter solutions through 0.22 μm filters before use. Consider adding 0.1% BSA as a carrier protein for very dilute solutions to prevent adsorption to surfaces.

• Challenge: Proteolytic degradation during storage or experimental procedures.
  Solution: Store lyophilized peptide at -20°C or -80°C in sealed containers with desiccant . For solutions, add protease inhibitor cocktails, aliquot to minimize freeze-thaw cycles, and store at -80°C. Consider regular purity analysis by HPLC or MS to monitor stability over time.

Experimental design considerations:

• Challenge: Inconsistent bioactivity results between experiments.
  Solution: Include internal standards in each assay run, standardize experimental conditions (temperature, pH, ionic strength), and validate activity relative to reference peptides. Implement standard curves with a commercial reference peptide preparation in each experiment.

• Challenge: Receptor desensitization in functional assays affecting reproducibility.
  Solution: Include appropriate wash-out periods between applications, use pulse rather than continuous application protocols, and consider pre-incubation with phosphatase inhibitors to slow desensitization mechanisms if studying prolonged signaling events.

Expression and purification obstacles:

• Challenge: Low expression yield in recombinant systems.
  Solution: Optimize codon usage for the expression host, consider fusion partners to enhance solubility, and systematically test induction conditions (temperature, inducer concentration, duration) . For E. coli systems, lower induction temperatures (16-25°C) often improve yield of correctly folded peptides.

• Challenge: Difficulty achieving high purity.
  Solution: Implement multi-step purification strategies combining orthogonal separation techniques (e.g., IMAC followed by ion exchange and RP-HPLC). Optimize chromatography conditions specifically for the peptide's physicochemical properties to achieve ≥85% purity as determined by SDS-PAGE .

These practical solutions address the most frequently encountered technical challenges while working with recombinant periviscerokinin peptides, enabling more consistent and reliable experimental outcomes.

How can researchers resolve data inconsistencies when comparing Periviscerokinin-3 activity across different experimental models and assay systems? [Advanced]

Resolving data inconsistencies when studying Periviscerokinin-3 across different experimental platforms requires systematic methodology standardization and advanced analytical approaches:

Standardization of peptide preparations:
Seemingly identical periviscerokinin-3 preparations can exhibit different activities due to subtle variations in:

• Post-translational modifications: High-resolution mass spectrometry with electron-transfer dissociation (ETD) fragmentation can detect subtle modifications like deamidation, oxidation, or truncation that may not be apparent in routine quality control but significantly impact activity.

• Conformational heterogeneity: Circular dichroism spectroscopy coupled with size-exclusion chromatography can identify differences in secondary structure or oligomerization state between preparations that may explain functional discrepancies.

• Reference standard establishment: Development of an absolutely quantified reference standard with defined bioactivity across multiple assay systems provides a crucial calibration tool for normalizing data across laboratories and experimental platforms.

Assay system normalization strategies:
Different assay systems inherently measure distinct aspects of periviscerokinin-3 activity. Reconciling these differences requires:

Advanced analytical frameworks for data integration:

• Operational models of agonism: Fitting concentration-response data to operational models (e.g., Black and Leff model) extracts system-independent parameters like efficacy (τ) and functional affinity (KA) that can be compared across diverse experimental systems.

• Bias factor calculation: Quantitative comparison of signaling pathway activation using the ΔΔlog(τ/KA) method accounts for system and observation bias, allowing true ligand bias to be distinguished from experimental artifacts.

• Bayesian meta-analysis: When multiple datasets show discrepancies, Bayesian hierarchical modeling can integrate these diverse data sources while explicitly accounting for inter-laboratory variability, yielding consensus parameter estimates with appropriately calibrated uncertainty.

Case study approach to resolving specific inconsistencies:
When facing specific data inconsistencies, a systematic troubleshooting approach is recommended:

• Identify pattern of discrepancy: Determine whether inconsistencies follow systematic patterns (e.g., consistently different EC50 values between assay types) or appear random.

• Isolate critical variables: Through factorial experimental design, systematically vary one experimental parameter at a time (peptide batch, buffer composition, incubation time) to identify critical factors driving inconsistency.

• Develop corrective protocols: Based on identified critical variables, establish standardized protocols that minimize variability, potentially including:

  • Specialized handling procedures for the peptide

  • Precise timing for sample preparation and assay execution

  • Consistent data analysis workflows with validated software tools

This comprehensive approach transforms apparent data inconsistencies from obstacles into opportunities for deeper mechanistic understanding of periviscerokinin-3 pharmacology and biology.