Recombinant Gromphadorina portentosa Sulfakinin-1

What is Gromphadorina portentosa Sulfakinin-1 and how does it function within the insect neuroendocrine system?

Gromphadorina portentosa Sulfakinin-1 is a neuropeptide found in Madagascar hissing cockroaches that belongs to the sulfakinin family, structurally and functionally homologous to vertebrate cholecystokinin (CCK). Based on research with related cockroach neuropeptides, sulfakinins likely regulate feeding behavior, digestive enzyme secretion, and gut motility.

Research methodology for characterization typically involves:

• Immunohistochemical analysis of tissue distribution, particularly in brain-corpus cardiacum and midgut paraneurons

• ELISA-based quantification in various tissues and hemolymph

• Physiological assays measuring effects on digestive enzyme activities

Studies of neuropeptides in the American cockroach (Periplaneta americana) demonstrate that related molecules like short neuropeptide F (sNPF) significantly inhibit α-amylase, protease, and lipase activities during starvation . Similar methodological approaches would be valuable for investigating Sulfakinin-1 function in Gromphadorina portentosa.

What expression systems are most appropriate for producing functional recombinant Gromphadorina portentosa Sulfakinin-1?

The selection of expression systems depends on research objectives and required post-translational modifications. The following table compares suitable expression systems:

For initial expression studies, the pEXP5NT/TOPO vector system has been successfully used for cockroach allergen expression in E. coli . This system could serve as a starting point for Sulfakinin-1 expression, particularly for structural characterization studies not requiring post-translational modifications.

How can researchers confirm the structural integrity of recombinant Gromphadorina portentosa Sulfakinin-1?

Verification of structural integrity requires a multi-faceted analytical approach:

• Mass spectrometry (MALDI-TOF MS or ESI-MS) to confirm:

  • Exact molecular weight

  • Presence of post-translational modifications (particularly tyrosine sulfation)

  • C-terminal amidation status

• Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

• NMR spectroscopy for detailed three-dimensional structure determination

• Western blotting using anti-sulfakinin antibodies or epitope tag detection

• Functional bioassays comparing activity with synthesized native peptide

The validation approach should be tailored to research objectives, with structural integrity confirmed before proceeding to functional studies .

What are the key considerations for designing primers to clone the Gromphadorina portentosa Sulfakinin-1 gene?

Primer design for Sulfakinin-1 gene amplification requires careful consideration of several factors:

• Sequence conservation analysis:

  • Alignment of known sulfakinin sequences from related cockroach species

  • Identification of conserved regions for degenerate primer design

  • Analysis of cockroach expressed sequence tag (EST) libraries

• Optimal primer design parameters:

  • 18-25 nucleotides length

  • 40-60% GC content

  • Melting temperatures (Tm) within 2-5°C of each other

  • Addition of restriction sites with appropriate flanking sequences

  • Consideration of codon optimization for expression system

• Recommended amplification approach:

  • Initial PCR with degenerate primers

  • Nested PCR for specificity enhancement

  • Rapid Amplification of cDNA Ends (RACE) for complete sequence determination

The methodology employed for German cockroach allergen identification, using EST clone analysis to obtain cDNA sequences, provides a viable template for Sulfakinin-1 gene isolation .

What purification strategies maximize yield and activity of recombinant Gromphadorina portentosa Sulfakinin-1?

Effective purification of recombinant Sulfakinin-1 requires a strategic multi-step approach:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Glutathione affinity for GST-fusion proteins

• Tag removal:

  • Site-specific protease cleavage (TEV, Factor Xa, or PreScission)

  • Optimization of cleavage conditions to prevent peptide degradation

• Polishing steps:

  • Reversed-phase HPLC for hydrophobicity-based separation

  • Size exclusion chromatography for aggregation removal

  • Ion exchange chromatography for charge-based purification

• Activity preservation considerations:

  • Buffer optimization to prevent aggregation

  • Addition of protease inhibitors throughout purification

  • Lyophilization conditions that maintain structural integrity

Yield and purity should be monitored at each step using SDS-PAGE, Western blotting, and mass spectrometry to ensure the final product meets research requirements.

How does recombinant Gromphadorina portentosa Sulfakinin-1 affect digestive enzyme regulation in comparison to native peptide?

This complex research question requires rigorous experimental design:

• Experimental methodology:

  • Isolation of midgut tissue from Gromphadorina portentosa

  • Tissue incubation with concentration gradients (10⁻¹⁰-10⁻⁶ M) of:
    a) Recombinant Sulfakinin-1
    b) Synthetic native Sulfakinin-1
    c) Control peptides with modified sequences

  • Measurement of enzyme activities:
    a) α-amylase using starch or p-nitrophenyl substrates
    b) Proteases using azocasein or fluorogenic substrates
    c) Lipases using p-nitrophenyl palmitate

• Expected experimental outcomes:

  • Dose-dependent inhibition of digestive enzymes

  • Activity comparison between recombinant and native forms

  • Identification of critical structural elements for activity

Research with American cockroach shows that sNPF significantly inhibits digestive enzyme activities during starvation, with activity restored upon refeeding . Similar methodological approaches would be appropriate for investigating Sulfakinin-1 effects.

What molecular mechanisms underlie receptor binding and activation by Gromphadorina portentosa Sulfakinin-1?

Investigation of receptor mechanisms requires sophisticated molecular and cellular approaches:

• Receptor identification and characterization:

  • Homology cloning based on known sulfakinin receptors

  • Expression in heterologous systems (HEK293, CHO cells)

  • Pharmacological characterization using competitive binding assays

• Signaling pathway analysis:

  • G-protein coupling determination (Gq, Gs, Gi)

  • Calcium mobilization assays

  • cAMP measurement

  • ERK phosphorylation analysis

• Structure-activity relationship studies:

  • Alanine scanning mutagenesis of the peptide

  • Receptor mutant analysis for binding domain identification

  • Fluorescently labeled peptide for binding visualization

The mechanisms of neuropeptide-receptor interactions have been studied in other cockroach species, providing methodological frameworks that can be adapted for Sulfakinin-1 research .

How do post-translational modifications affect the bioactivity of recombinant Gromphadorina portentosa Sulfakinin-1?

Sulfakinins typically require specific post-translational modifications for full biological activity:

• Critical modifications to evaluate:

  • Tyrosine sulfation

  • C-terminal amidation

  • Disulfide bond formation (if applicable)

• Experimental design approach:

  • Production of variant peptides with different modification patterns

  • Comparative receptor binding assays

  • Functional bioassays (feeding regulation, gut motility)

  • Circular dichroism spectroscopy for structural comparison

• Expected functional differences:

  • Reduced receptor binding affinity for non-sulfated variants

  • Decreased signaling efficacy for non-amidated forms

  • Potential conformational changes affecting receptor recognition

This systematic approach allows for precise determination of structure-function relationships critical for understanding Sulfakinin-1 bioactivity .

What methodological approaches can identify cross-reactivity between Gromphadorina portentosa Sulfakinin-1 and sulfakinins from other species?

Cross-reactivity studies require multiple complementary approaches:

• Receptor pharmacology:

  • Heterologous expression of sulfakinin receptors from different species

  • Competitive binding assays with radiolabeled or fluorescent peptides

  • Calcium mobilization or cAMP assays to measure activation potency

  • Receptor internalization studies

• Immunological cross-reactivity:

  • Development of specific antibodies against Gromphadorina portentosa Sulfakinin-1

  • ELISA and Western blot analysis with sulfakinins from related species

  • Immunoabsorption studies to identify shared epitopes

  • Immunohistochemical tissue staining comparison across species

• Bioinformatic analysis:

  • Sequence alignment and phylogenetic analysis

  • Molecular modeling of peptide-receptor interactions

  • Prediction of structural conservation and divergence

Research on cockroach allergen cross-reactivity provides methodological guidance, where immunoblot and immunoblot inhibition studies effectively identified shared and unique epitopes between American and German cockroach allergens .

How can researchers design functional assays to evaluate the physiological effects of Gromphadorina portentosa Sulfakinin-1 in vivo?

Comprehensive functional characterization requires multiple physiological assays:

• Feeding behavior assays:

  • Precise food intake measurement following Sulfakinin-1 injection

  • Analysis of meal size, frequency, and duration

  • Two-choice preference tests to evaluate satiety effects

  • Dose-response relationships and temporal dynamics

• Digestive physiology:

  • In vivo measurement of digestive enzyme activities following peptide administration

  • Analysis of enzyme gene expression using quantitative RT-PCR

  • Food transit time using colored markers

  • Nutritional parameter analysis (hemolymph trehalose, lipids, amino acids)

• Electrophysiological approaches:

  • Isolated gut motility recording in organ bath preparations

  • Intracellular recordings from stomatogastric neurons

  • Multi-electrode array recordings from gut neural networks

Research with American cockroach demonstrated that injection of sNPF into the hemocoel led to decreased digestive enzyme activities, providing a methodological template for similar studies with Sulfakinin-1 .