Recombinant Supella dimidiata Periviscerokinin-3

What is Supella dimidiata Periviscerokinin-3 and how does it relate to other insect neuropeptides?

Supella dimidiata Periviscerokinin-3 is a member of the CAPA peptide family found in the brown-banded cockroach (Supella dimidiata). CAPA peptides, including periviscerokinin (PVK) variants, are distributed throughout major insect taxa, particularly within Dictyoptera (cockroaches and termites). These neuropeptides are typically expressed in perisympathetic organs (PSOs) and function as signaling molecules in various physiological processes.

Most cockroach species express three different PVKs (PVK-1, PVK-2, and PVK-3) and a single CAPA-pyrokinin (PK), although certain species like Cryptocercus and some blattellid cockroaches express only two different PVKs. Some Madagascan Blaberidae and other species express a fourth PVK variant (PVK-4) . The specific sequence of Supella dimidiata PVK-3 can be determined through tandem mass spectrometry, allowing for comparative analysis with other insect neuropeptides.

What biological functions does Periviscerokinin-3 serve in insect physiology?

CAPA peptides, including PVK-3, function as neuropeptides involved in regulating various physiological processes in insects. These include:

• Myotropic activity (affecting muscle contractions)

• Diuretic and antidiuretic functions (regulating water balance)

• Cardioacceleratory effects

• Modulation of ion transport across epithelia

PVK-3 specifically contributes to these regulatory mechanisms, with its conserved sequence motifs playing critical roles in receptor binding and subsequent signal transduction. The conservation of these peptides across diverse insect taxa indicates their fundamental importance in insect neurophysiology and homeostasis .

How conserved is PVK-3 across different cockroach species?

Mass spectrometric analysis of abdominal PSO preparations from various cockroach species reveals that CAPA peptides, including PVK-3, show significant conservation in their core sequences while exhibiting species-specific variations. These variations make them valuable markers for phylogenetic studies.

The sequence conservation is particularly evident within closely related taxa, while more distant relationships show greater sequence divergence. This pattern of conservation and variation allows researchers to use PVK-3 sequences to complement molecular biological and morphological data for reconstructing phylogenetic relationships across insect taxa .

What is the optimal expression system for producing recombinant Supella dimidiata PVK-3?

The optimal expression system for recombinant Supella dimidiata PVK-3 depends on research objectives, but several systems have proven effective for insect neuropeptides:

For most research applications, E. coli remains the preferred system when using a fusion partner (such as thioredoxin, SUMO, or MBP) to enhance solubility and facilitate purification. Codon optimization for E. coli expression is essential when expressing insect peptides to overcome codon bias issues .

What purification strategies are most effective for isolating recombinant PVK-3 with high purity?

Purification of recombinant PVK-3 typically involves a multi-step approach:

• Initial Capture: Affinity chromatography using a fusion tag (His-tag, GST, etc.)

• Fusion Protein Cleavage: Using a specific protease (TEV, thrombin, etc.) to release the target peptide

• Secondary Purification: Reverse-phase HPLC to separate the target peptide from the fusion partner and impurities

• Final Polishing: Size exclusion chromatography or ion-exchange chromatography

For analytical verification of the purified peptide, mass spectrometry techniques (similar to those used for native peptide sequencing) can confirm the exact mass and sequence of the recombinant product. Tandem mass spectrometry is particularly valuable for sequence confirmation, as demonstrated in the analysis of native CAPA peptides from various insect species .

How can mass spectrometry techniques be optimized for characterizing recombinant PVK-3?

Mass spectrometry plays a crucial role in characterizing both native and recombinant PVK-3. Based on approaches used for native CAPA peptides:

• Sample Preparation: Direct preparation of abdominal PSO or purified recombinant peptide

• Ionization Method: Matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI)

• Analysis Mode: Tandem mass spectrometry (MS/MS) for sequence determination

• Fragmentation: Collision-induced dissociation (CID) or electron-transfer dissociation (ETD)

For recombinant PVK-3, the mass spectrometric approach should include:

• Accurate mass determination to confirm proper synthesis

• Fragmentation pattern analysis to verify the amino acid sequence

• Comparison with theoretical and native peptide spectra

These techniques can reveal complete sequences and post-translational modifications, allowing for accurate structure determination as demonstrated in the analysis of CAPA peptides from 61 cockroach/termite species .

How can recombinant PVK-3 be utilized in phylogenetic studies of Blattodea?

Recombinant PVK-3 can serve as a valuable tool in phylogenetic studies through several approaches:

• Reference Material: Providing authenticated standards for mass spectrometric identification of native peptides

• Sequence Verification: Confirming putative sequences derived from genomic or transcriptomic data

• Antibody Production: Generating specific antibodies for immunohistochemical detection across species

• Structure-Function Analysis: Determining conserved motifs essential for biological activity

The sequences of CAPA peptides, including PVK-3, have been successfully used to complement molecular, biological, and morphological data for phylogenetic analysis. Studies incorporating 61 species have shown that neuropeptide-based cladograms generally agree with recent molecular and morphological phylogenetic analyses, including the placement of termites within cockroaches .

What structure-activity relationship studies can be conducted using recombinant PVK-3 variants?

Structure-activity relationship (SAR) studies using recombinant PVK-3 variants can elucidate the functional importance of specific amino acid residues. These studies typically involve:

• Alanine Scanning: Systematic replacement of each amino acid with alanine to identify essential residues

• Conservative/Non-conservative Substitutions: Replacing residues with similar or dissimilar amino acids

• N/C-terminal Truncations: Determining minimal active sequence

• Cyclization/Conformational Constraints: Exploring the role of peptide backbone flexibility

The results from such studies can reveal:

• Critical residues for receptor binding

• Residues involved in signaling activation

• Structural elements that confer selectivity among receptor subtypes

• Peptide regions that can be modified to enhance stability without affecting activity

These insights can guide the development of more stable analogs or selective receptor agonists/antagonists for further physiological studies.

How do post-translational modifications affect the activity of recombinant PVK-3 compared to native peptides?

Post-translational modifications (PTMs) can significantly influence the biological activity of neuropeptides. For PVK-3, critical considerations include:

When comparing recombinant and native PVK-3, researchers should account for these modifications, as their absence in recombinant peptides produced in E. coli can lead to reduced bioactivity. Mass spectrometric techniques similar to those used in the phylogenetic studies of CAPA peptides can verify the presence or absence of these modifications .

What are the main challenges in confirming the correct folding of recombinant PVK-3?

While short peptides like PVK-3 typically have limited tertiary structure, confirming proper folding remains important for biological activity. Key challenges and solutions include:

• Limited Structural Elements: PVK-3, being a relatively short peptide, may not have extensive secondary structure, making traditional structural biology techniques less informative

• Potential for Aggregation: Recombinant neuropeptides can form aggregates that reduce activity

• Critical C-terminal Amidation: Ensuring proper C-terminal processing is essential for activity

Analytical approaches to address these challenges:

• Circular Dichroism (CD) spectroscopy to assess secondary structure elements

• Size Exclusion Chromatography to detect aggregation

• Functional bioassays comparing recombinant peptide activity to synthetic standards

• NMR spectroscopy for detailed structural characterization

• Mass spectrometry to confirm modifications and proper processing

The analytical techniques used for CAPA peptide characterization in phylogenetic studies provide a foundation for these approaches .

How can researchers troubleshoot poor yields of recombinant PVK-3 expression?

Poor yields of recombinant PVK-3 can stem from various factors. Systematic troubleshooting should consider:

• Expression Vector Design:

  • Optimize codon usage for the expression host

  • Include fusion partners known to enhance peptide solubility (MBP, SUMO, thioredoxin)

  • Consider dual fusion systems for very difficult peptides

• Expression Conditions:

  • Test multiple temperatures (18°C, 25°C, 37°C)

  • Vary induction parameters (IPTG concentration, induction time)

  • Screen different media formulations

• Host Cell Selection:

  • Compare different E. coli strains (BL21(DE3), Rosetta, SHuffle)

  • Consider alternative expression systems for problematic peptides

• Purification Optimization:

  • Adjust lysis conditions to maximize peptide recovery

  • Optimize buffer compositions to enhance stability

  • Evaluate different chromatography techniques

A systematic approach to these parameters can significantly improve recombinant peptide yields, allowing for better characterization and functional studies.

What are the best approaches for developing sensitive and specific assays to detect PVK-3 activity?

Developing sensitive and specific assays for PVK-3 activity requires consideration of its physiological functions. Recommended approaches include:

• Receptor Binding Assays:

  • Heterologous expression of PVK receptors in cell lines

  • Competitive binding assays using labeled PVK-3

  • BRET/FRET-based assays for real-time interaction monitoring

• Functional Assays:

  • Calcium mobilization assays in receptor-expressing cells

  • cAMP or IP3 measurement for second messenger detection

  • Myotropic assays using isolated insect tissue (e.g., hindgut preparations)

• Ex Vivo Physiological Measurements:

  • Fluid secretion assays using isolated Malpighian tubules

  • Muscle contraction measurements in isolated gut preparations

  • Electrophysiological recordings of neuronal activity

• ELISA-Based Detection:

  • Development of specific antibodies against PVK-3

  • Competitive ELISA for quantification in biological samples

These assays can be calibrated using native peptides isolated from cockroach species, which can be sequenced using mass spectrometry techniques similar to those employed in phylogenetic studies of CAPA peptides .

How can recombinant PVK-3 contribute to understanding evolutionary relationships among Blattodea?

Recombinant PVK-3 can significantly enhance evolutionary studies of Blattodea through:

• Reference Standards for Native Peptide Identification:

  • Providing authenticated standards for mass spectrometric analysis

  • Facilitating high-throughput screening of multiple species

• Sequence Conservation Analysis:

  • Comparing PVK-3 sequences across diverse cockroach species

  • Identifying conserved motifs versus variable regions that reflect evolutionary distance

• Combined Peptidic Datasets:

  • Integrating PVK-3 sequence data with other neuropeptide families (e.g., adipokinetic hormones, sulfakinins)

  • Generating more robust phylogenetic trees with improved bootstrap values

Research has demonstrated that neuropeptide sequences can effectively complement molecular biological and morphological data for phylogenetic reconstruction. Cladograms generated from peptide sequences align with recent molecular and morphological analyses, including the phylogenetic arrangement placing termites within cockroaches .

What experimental approaches can determine PVK-3 receptor specificity across different insect species?

Understanding PVK-3 receptor specificity across different insect species requires:

• Receptor Cloning and Expression:

  • Identifying putative receptors through genomic/transcriptomic analysis

  • Heterologous expression in cell lines (HEK293, CHO, Sf9)

• Cross-Species Activation Profiles:

  • Testing recombinant PVK-3 from multiple species against receptors from different species

  • Determining EC50 values to quantify specificity differences

• Receptor Pharmacology:

  • Competitive binding assays to measure receptor affinity

  • Functional assays (calcium mobilization, cAMP) to measure receptor activation

  • Bioinformatic analysis of receptor sequences to identify critical binding domains

• In vivo Validation:

  • Testing cross-species responses in live insects

  • CRISPR-mediated receptor modification to confirm specificity determinants

This systematic approach can reveal how receptor-ligand co-evolution has shaped the diverse functions of PVK-3 across the Blattodea order and other insect groups.

How do the structure and function of recombinant PVK-3 compare with synthetic analogues?

Comparing recombinant PVK-3 with synthetic analogues provides insights into production methods and structural requirements:

Key considerations for functional comparisons:

• Ensuring C-terminal amidation in both preparations

• Controlling for potential contaminants from the expression system

• Using multiple biological assays to comprehensively assess activity

Tandem mass spectrometry, similar to the approach used for sequencing native CAPA peptides from cockroaches and termites, can verify structural equivalence between recombinant and synthetic peptides .

What are the latest mass spectrometric techniques for characterizing PVK variants in different insect species?

Advanced mass spectrometric techniques have revolutionized the characterization of PVKs across insect species:

• Direct Analysis Techniques:

  • Direct tissue analysis using MALDI-TOF MS

  • Single-cell mass spectrometry for neuron-specific peptide profiling

  • Imaging mass spectrometry for spatial distribution of PVKs

• High-Sensitivity Detection:

  • Nano-LC coupled to MS/MS for detection of low-abundance variants

  • Multiple reaction monitoring (MRM) for targeted quantification

  • Ion mobility separation for enhanced isomer discrimination

• Structural Characterization:

  • Top-down proteomics approaches for intact peptide analysis

  • Electron-transfer dissociation (ETD) for improved sequence coverage

  • High-resolution accurate mass (HRAM) instruments for unambiguous identification

These techniques have enabled the complete sequencing of CAPA peptides from single specimens across 61 cockroach/termite species, demonstrating their utility in comprehensive peptide characterization for phylogenetic studies .

How can genomic and transcriptomic approaches complement proteomic studies of PVK-3?

Integrating genomic and transcriptomic approaches with proteomic studies provides a comprehensive understanding of PVK-3:

• Genomic Analysis:

  • Identification of PVK gene structure and regulatory elements

  • Comparative genomics to trace evolutionary history

  • Identification of gene duplication events (explaining multiple PVK variants)

• Transcriptomic Approaches:

  • RNA-Seq to confirm gene expression in specific tissues

  • Quantitative PCR for expression level comparison across developmental stages

  • Single-cell transcriptomics to identify PVK-producing neurons

• Integration with Proteomics:

  • Confirmation of predicted peptide sequences through MS/MS

  • Identification of post-translational modifications

  • Validation of alternative splicing events

• Functional Validation:

  • CRISPR/Cas9 genome editing to study PVK gene function

  • RNAi to investigate phenotypic effects of PVK knockdown

The complementary nature of these approaches provides robust evidence for PVK sequence determination, expression patterns, and functional roles, enhancing the value of neuropeptide data for phylogenetic analyses .

What bioinformatic tools are most effective for analyzing evolutionary relationships based on neuropeptide sequences?

Several bioinformatic approaches are particularly valuable for neuropeptide-based phylogenetic analysis:

• Sequence Alignment Tools:

  • MUSCLE or MAFFT for multiple sequence alignment of short peptides

  • Manual curation to ensure proper alignment of conserved motifs

  • PRALINE for alignment incorporating secondary structure predictions

• Phylogenetic Analysis Methods:

  • Maximum Parsimony for direct sequence comparison

  • Bayesian Inference for probability-based tree construction

  • Maximum Likelihood methods with appropriate substitution models

• Specialized Neuropeptide Resources:

  • DINeR (Database of Insect Neuropeptide Research)

  • NeuroPred for predicting neuropeptide cleavage sites

  • PepBank for comparative analysis of bioactive peptides

• Combined Data Analysis:

  • Tools for integrating peptide data with molecular and morphological characters

  • Bootstrap analysis to assess tree reliability

  • Consensus tree methods to resolve conflicts between different data types

These approaches have successfully demonstrated that neuropeptide sequences can complement molecular and morphological data for phylogenetic reconstruction, with improved bootstrap values when multiple neuropeptide families are analyzed together .

How can researchers address data inconsistencies when comparing PVK sequences across different analytical platforms?

Researchers may encounter discrepancies when comparing PVK sequences characterized using different analytical approaches. Strategies to address these inconsistencies include:

• Standardization of Analytical Methods:

  • Establish common mass spectrometry parameters across laboratories

  • Develop reference standards for instrument calibration

  • Create publicly available spectral libraries for PVK peptides

• Cross-Validation Approaches:

  • Confirm MS/MS sequence assignments using synthetic peptides

  • Validate mass spectrometry findings with Edman sequencing when possible

  • Compare direct tissue analysis with peptide extracts to identify preparation artifacts

• Data Integration Protocols:

  • Develop algorithms to normalize data from different platforms

  • Establish confidence scores for sequence assignments

  • Implement decision trees for resolving conflicting results

• Reporting Standards:

  • Document complete methodological details

  • Report raw data alongside interpreted sequences

  • Include quality control metrics with published sequences

These approaches can minimize discrepancies that might arise when comparing sequences determined by different laboratories or analytical platforms, enhancing the reliability of neuropeptide data for phylogenetic studies .

What strategies can optimize the stability of recombinant PVK-3 during purification and storage?

Optimizing stability of recombinant PVK-3 requires attention to several factors:

• Buffer Optimization:

  • pH screening (typically 5.0-7.5) to identify optimal stability range

  • Ionic strength optimization to minimize aggregation

  • Addition of stabilizing excipients (glycerol, trehalose, albumin)

• Temperature Considerations:

  • Short-term storage at 4°C with preservatives

  • Long-term storage at -80°C in single-use aliquots

  • Lyophilization for extended shelf-life

• Chemical Stability Enhancements:

  • Minimizing oxidation through addition of reducing agents

  • Preventing peptide bond hydrolysis by avoiding extreme pH

  • Inhibiting proteolytic degradation with protease inhibitors

• Formulation Strategies:

  • Development of stabilized liquid formulations

  • Optimization of freeze-drying protocols

  • Controlled rate freezing to minimize freeze-thaw damage

These approaches can significantly extend the usable life of recombinant PVK-3 preparations, ensuring consistent results across extended research projects.

How can researchers distinguish between technical artifacts and true structural variants when analyzing PVK-3?

Distinguishing between technical artifacts and true structural variants is crucial for accurate PVK-3 characterization:

• Common Technical Artifacts:

  • Deamidation of asparagine/glutamine residues (mass shift +0.984 Da)

  • Oxidation of methionine/tryptophan (+15.995 Da)

  • Carbamylation of N-termini/lysines (+43.006 Da)

  • Sodium/potassium adducts (+21.982/+38.095 Da)

• Validation Strategies:

  • Multiple fragmentation techniques (CID, ETD, HCD) for sequence confirmation

  • Analysis of isotopic patterns to distinguish modifications from isotope peaks

  • Comparison of retention times with synthetic standards

  • Multiple biological and technical replicates to identify consistent features

• Computational Approaches:

  • De novo sequencing algorithms with manual verification

  • Database searching with expanded modification parameters

  • Statistical approaches to distinguish noise from signal

• Biological Validation:

  • Genomic/transcriptomic confirmation of variants

  • Evolutionary context analysis (comparing closely related species)

  • Functional testing of putative variants

These approaches can help researchers confidently identify true structural variants of PVK-3 across different species, enhancing the reliability of peptide-based phylogenetic analyses .