Recombinant Torpedo marmorata Des[Ser (1)Pro (2)] scyliorhinin-2

What is the structural relationship between scyliorhinin-2 and other tachykinins?

Scyliorhinin-2 belongs to the tachykinin family of peptides first isolated from dogfish intestinal tissue. The native scyliorhinin-2 from Scyliorhinus caniculus has the amino acid sequence Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met-NH2. This peptide shares functional similarity with mammalian tachykinins in its ability to contract guinea pig ileal longitudinal muscle, though it differs structurally from substance P and neurokinin A, which are absent in dogfish intestinal tissue . The Des[Ser (1)Pro (2)] modification refers to the removal of the first two N-terminal amino acids (Serine and Proline), potentially altering receptor binding properties while preserving the critical C-terminal region that is characteristic of the tachykinin family.

What analytical methods are most effective for confirming the identity of recombinant Des[Ser (1)Pro (2)] scyliorhinin-2?

Multiple complementary analytical techniques should be employed to verify the identity of recombinant Des[Ser (1)Pro (2)] scyliorhinin-2. Mass spectrometry provides precise molecular weight determination, which for the truncated peptide would correspond to the theoretical mass minus the first two amino acids. Edman degradation confirms the N-terminal sequence, which should begin with Ser in position 3 of the native sequence. Reversed-phase HPLC paired with UV detection at 214 nm and 280 nm can establish purity and comparison with reference standards. Similar verification approaches have been successfully employed for other recombinant proteins, such as the N-terminal fragments of Torpedo californica acetylcholine receptor α-subunits expressed in Escherichia coli and verified through Edman degradation and mass spectrometry .

What expression systems are optimal for producing functional Des[Ser (1)Pro (2)] scyliorhinin-2?

For recombinant production of Des[Ser (1)Pro (2)] scyliorhinin-2, bacterial expression systems using Escherichia coli represent a well-established and cost-effective approach. Two strategic vector designs should be considered: either incorporating a His-tag preceding the peptide sequence or following it, similar to the approach used for expressing Torpedo acetylcholine receptor fragments . For small peptides like modified scyliorhinin-2, researchers may consider using fusion protein systems with cleavable tags such as GST, SUMO, or thioredoxin to enhance expression yields and solubility. Purification through Ni²⁺-agarose affinity chromatography, followed by tag removal via specific proteases and further purification steps such as reverse-phase HPLC, provides the highest purity. Alternative eukaryotic expression systems, including yeast or mammalian cells, may be considered if post-translational modifications are required.

How can researchers optimize codon usage for expressing Des[Ser (1)Pro (2)] scyliorhinin-2 in heterologous systems?

Codon optimization represents a critical consideration when expressing elasmobranch-derived peptides in heterologous systems. For recombinant Des[Ser (1)Pro (2)] scyliorhinin-2 expression, researchers should:

• Analyze codon usage tables for the target expression host (e.g., E. coli BL21(DE3) or Rosetta strains)

• Replace rare codons with synonymous high-frequency codons of the expression host

• Balance GC content (typically 40-60%) to enhance mRNA stability

• Avoid secondary structures in mRNA that might impede translation

• Consider using specialized strains supplemented with rare tRNAs if full codon optimization is impractical

This approach has proven successful in optimizing expression of other marine-derived peptides and can significantly increase yield from 2-10 fold.

What purification challenges are specific to Des[Ser (1)Pro (2)] scyliorhinin-2 and how can they be addressed?

Purification of Des[Ser (1)Pro (2)] scyliorhinin-2 presents several challenges related to its cysteine content and amphipathic nature. Researchers should implement the following methodological approaches:

The presence of two cysteine residues in the truncated peptide necessitates careful control of redox conditions during purification to prevent formation of incorrect disulfide bonds or aggregation. Purification under reducing conditions with 1-5 mM DTT or 2-mercaptoethanol, followed by controlled oxidative refolding, is recommended. Affinity chromatography using immobilized metal affinity chromatography (IMAC) with Ni²⁺-agarose columns can be employed for His-tagged constructs, similar to the approach used for Torpedo acetylcholine receptor fragments . Subsequently, reverse-phase HPLC should be used for final purification, with careful optimization of acetonitrile gradients (typically 15-40%) in 0.1% TFA to achieve >95% purity.

What are the preferred methodologies for assessing tachykinin receptor binding of Des[Ser (1)Pro (2)] scyliorhinin-2?

Assessment of Des[Ser (1)Pro (2)] scyliorhinin-2 binding to tachykinin receptors should employ competitive binding assays using radiolabeled tachykinins. The methodological approach should include:

• Preparation of membrane fractions from tissues expressing tachykinin receptors or from recombinant cells expressing specific receptor subtypes

• Competitive displacement of a radiolabeled reference ligand (e.g., [¹²⁵I]substance P or [³H]substance P) by increasing concentrations of Des[Ser (1)Pro (2)] scyliorhinin-2

• Determination of IC₅₀ values and calculation of binding affinity (Ki) using the Cheng-Prusoff equation

• Comparative analysis with native scyliorhinin-2 and other tachykinins to establish receptor subtype selectivity profiles

This approach allows quantitative assessment of how the N-terminal truncation affects receptor binding properties, similar to binding studies performed with [¹²⁵I]α-bungarotoxin for acetylcholine receptor fragments, which yielded KD values of approximately 130 nM .

How should contractile assays be designed to evaluate Des[Ser (1)Pro (2)] scyliorhinin-2 biological activity?

Contractile assays represent the gold standard for functional characterization of tachykinins and should be designed as follows for Des[Ser (1)Pro (2)] scyliorhinin-2:

Isolated longitudinal muscle strips from guinea pig ileum should be mounted in organ baths containing oxygenated Krebs solution at 37°C. Cumulative concentration-response curves (10⁻¹⁰ to 10⁻⁶ M) should be generated for Des[Ser (1)Pro (2)] scyliorhinin-2 with 5-minute intervals between successive applications. Control experiments with native scyliorhinin-2 should be performed in parallel tissues to generate comparative EC₅₀ values and maximal responses. Specificity can be confirmed through tachykinin receptor antagonists (e.g., SR140333 for NK1, SR48968 for NK2).

Data should be analyzed using non-linear regression to determine potency (pEC₅₀) and efficacy (Emax) values. The contractile profile of scyliorhinin-2 would be compared to its activity in dogfish intestine, reflecting the biological activity described in the original characterization of scyliorhinin-1 and scyliorhinin-2 .

What cellular signaling pathways are activated by Des[Ser (1)Pro (2)] scyliorhinin-2 and how can they be measured?

Des[Ser (1)Pro (2)] scyliorhinin-2 likely activates G-protein coupled receptor signaling pathways typical of tachykinins. To comprehensively characterize these pathways, researchers should implement:

• Calcium mobilization assays using fluorescent indicators (Fura-2/AM or Fluo-4) in receptor-expressing cells to measure intracellular Ca²⁺ transients

• Phospholipase C activation measurements through quantification of inositol phosphates

• ERK1/2 phosphorylation analysis via western blotting with phospho-specific antibodies

• cAMP measurements to assess potential coupling to Gs or Gi proteins

• β-arrestin recruitment assays to evaluate receptor desensitization pathways

Comparison of signaling profiles between native and truncated peptides can reveal biased agonism properties - where certain pathways are preferentially activated over others. Time-course experiments (0-60 minutes) should be conducted to characterize the kinetics of pathway activation, with EC₅₀ values determined for each signaling endpoint to construct a comprehensive pharmacological profile.

How does Des[Ser (1)Pro (2)] scyliorhinin-2 from Torpedo marmorata compare to tachykinins from other elasmobranch species?

Comparative analysis of Des[Ser (1)Pro (2)] scyliorhinin-2 from Torpedo marmorata with tachykinins from other elasmobranchs provides evolutionary insights into peptide diversification. While scyliorhinin-2 was originally identified in Scyliorhinus caniculus with the sequence Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met-NH2 , researchers should perform:

• Sequence alignment of tachykinins across elasmobranch species using multiple sequence alignment tools

• Phylogenetic analysis to establish evolutionary relationships

• Functional comparisons through standardized bioassays

• Receptor binding profiles across species to identify conserved pharmacophores

Of particular interest is the comparison between dogfish (Scyliorhinus) and electric ray (Torpedo) derived peptides, as these represent distinct elasmobranch lineages. The absence of substance P and neurokinin A in dogfish intestinal tissue suggests unique evolutionary adaptations in the tachykinin system of elasmobranchs compared to mammals. This comparative approach illuminates how sequence variations influence receptor subtype selectivity and provides insights into the evolution of neuropeptide-receptor systems.

What is the evolutionary significance of tachykinin diversity in elasmobranchs?

The diversity of tachykinins in elasmobranchs, including variants like Des[Ser (1)Pro (2)] scyliorhinin-2, represents an important evolutionary model for understanding neuropeptide diversification. Methodologically, researchers should:

• Conduct comprehensive genomic and transcriptomic analyses across elasmobranch species, similar to those performed for the small-spotted catshark Scyliorhinus canicula

• Identify tachykinin gene loci and compare gene structures, focusing on exon-intron boundaries

• Analyze conserved regulatory elements that might influence tissue-specific expression

• Examine post-translational processing pathways across species

The presence of unique tachykinins in elasmobranchs while mammalian counterparts (substance P and neurokinin A) are absent suggests evolutionary divergence in neuropeptide systems. This divergence may reflect adaptation to specific physiological demands in the elasmobranch lineage. Understanding this diversity provides insights into the co-evolution of neuropeptides and their receptors, potentially revealing novel biological functions that evolved independently in different vertebrate lineages.

How do the receptor binding properties of Des[Ser (1)Pro (2)] scyliorhinin-2 differ across vertebrate species?

The receptor binding properties of Des[Ser (1)Pro (2)] scyliorhinin-2 likely vary significantly across vertebrate species due to evolutionary divergence in tachykinin receptor structure. To systematically characterize these differences, researchers should:

• Express recombinant tachykinin receptors (NK1, NK2, NK3) from different vertebrate species (elasmobranchs, teleosts, amphibians, mammals) in a common cellular background

• Perform comparative radioligand binding assays with consistent methodology

• Generate comprehensive affinity profiles (pKi values) across receptor subtypes and species

• Correlate binding affinities with receptor sequence differences to identify critical determinants of ligand recognition

This cross-species approach reveals evolutionary trends in peptide-receptor co-evolution and identifies conserved binding motifs. The data can be presented as a comprehensive affinity matrix showing receptor subtype selectivity across phylogeny:

How can Des[Ser (1)Pro (2)] scyliorhinin-2 be used as a tool for investigating tachykinin receptor subtypes?

Des[Ser (1)Pro (2)] scyliorhinin-2 represents a valuable pharmacological tool for dissecting tachykinin receptor subtype functions. Methodologically, researchers should:

• Perform comprehensive radioligand binding assays across all tachykinin receptor subtypes to establish selectivity profiles

• Create fluorescently labeled derivatives by conjugating fluorophores at specific positions away from the pharmacophore

• Develop covalent photoaffinity analogs by incorporating photoreactive groups for receptor labeling

• Utilize the peptide in receptor mutagenesis studies to identify critical receptor-ligand interaction points

• Employ the modified peptide in competition binding studies to characterize novel tachykinin ligands

The N-terminal truncation potentially confers unique receptor subtype selectivity, making Des[Ser (1)Pro (2)] scyliorhinin-2 particularly valuable for distinguishing between closely related receptor subtypes. Researchers can exploit these properties to develop subtype-selective agonists and antagonists, similar to approaches used with other receptor systems like the acetylcholine receptor .

What are the challenges in developing stable isotope-labeled Des[Ser (1)Pro (2)] scyliorhinin-2 for structural studies?

Development of stable isotope-labeled Des[Ser (1)Pro (2)] scyliorhinin-2 for NMR and other structural studies presents several methodological challenges that must be addressed:

For recombinant expression of ¹⁵N/¹³C-labeled peptide, researchers should culture bacteria in minimal media containing ¹⁵NH₄Cl as the sole nitrogen source and ¹³C-glucose as the carbon source. Expression yield optimization requires careful temperature control (typically 16-25°C) and IPTG concentration adjustment (0.1-1.0 mM). For peptides with disulfide bonds like scyliorhinin-2, proper folding must be verified through circular dichroism and native PAGE.

Site-specific labeling can be achieved through expressed protein ligation or selective amino acid type labeling. Purification of labeled peptides follows standard protocols but requires validation of isotope incorporation through mass spectrometry. Typical incorporation rates should exceed 95% for high-quality NMR studies. The labeled peptide enables detailed structural studies including backbone dynamics and receptor-binding conformational changes using multi-dimensional NMR experiments (HSQC, NOESY, TOCSY).

What experimental approaches can resolve contradictory data on Des[Ser (1)Pro (2)] scyliorhinin-2 biological activity?

When confronted with contradictory data regarding Des[Ser (1)Pro (2)] scyliorhinin-2 biological activity, researchers should implement a systematic troubleshooting approach:

• Batch-to-batch consistency verification through analytical HPLC, mass spectrometry, and circular dichroism to ensure identity and proper folding

• Standardization of experimental protocols across laboratories using identical buffer compositions, tissue preparations, and data analysis methods

• Blind testing of samples by independent laboratories to eliminate bias

• Comprehensive receptor profiling against all known tachykinin receptors and related GPCRs to identify potential off-target activities

• Evaluation of peptide stability under experimental conditions using LC-MS time-course analysis

Investigation of potential species-specific effects is critical, as receptor pharmacology can vary significantly between model systems. Contradictory functional data may result from differences in receptor expression levels, G-protein coupling efficiency, or downstream signaling components between experimental systems. Researchers should systematically document these variables and establish standardized positive controls (e.g., substance P) to normalize responses across different experimental platforms.

What storage conditions ensure long-term stability of Des[Ser (1)Pro (2)] scyliorhinin-2?

Maintaining the stability of Des[Ser (1)Pro (2)] scyliorhinin-2 requires careful attention to storage conditions due to its susceptibility to oxidation and aggregation. Researchers should implement the following methodological approach:

Lyophilized peptide should be stored at -20°C or preferably -80°C under inert gas (nitrogen or argon) to prevent oxidation of the sensitive methionine residue at the C-terminus and cysteine residues. For reconstituted peptide, storage in small single-use aliquots (50-100 μl) at concentrations of 0.1-1.0 mg/ml in 0.1% TFA or 10 mM acetic acid at -80°C minimizes freeze-thaw cycles. Inclusion of 5-10% acetonitrile may enhance solubility without compromising stability.

Stability should be verified through periodic analytical HPLC and mass spectrometry to detect degradation products or aggregation. Typical shelf-life under optimal storage conditions is 12-24 months for lyophilized material and 3-6 months for reconstituted peptide. Addition of reducing agents (e.g., 1 mM DTT) may be necessary if the peptide contains free cysteine residues to prevent disulfide bond formation and aggregation.

How can researchers troubleshoot inconsistent results in receptor binding studies with Des[Ser (1)Pro (2)] scyliorhinin-2?

Inconsistent receptor binding results with Des[Ser (1)Pro (2)] scyliorhinin-2 can stem from multiple sources. A systematic troubleshooting approach includes:

• Peptide quality verification through analytical HPLC (>95% purity) and mass spectrometry to confirm identity

• Fresh preparation of stock solutions in appropriate vehicles (water with 10% DMSO or 10% acetonitrile) to ensure solubility

• Verification of receptor expression levels in membrane preparations through saturation binding assays

• Standardization of binding assay conditions (buffer composition, pH, temperature, incubation time)

• Implementation of positive controls (known tachykinin ligands) in each experiment

For competitive binding assays, researchers should ensure that radioligand concentration is below KD (typically 0.1-0.5×KD) and that non-specific binding is properly defined using high concentrations (1-10 μM) of unlabeled ligand. Signal-to-noise ratios should exceed 5:1 for reliable data. Assay window optimization may require adjustment of membrane protein concentration (typically 10-50 μg/ml). Data analysis should employ consistent mathematical models (one-site vs. two-site competition) across experiments to ensure comparability of affinity constants.

What quality control parameters should be established for recombinant Des[Ser (1)Pro (2)] scyliorhinin-2 production?

Rigorous quality control for recombinant Des[Ser (1)Pro (2)] scyliorhinin-2 is essential for research reproducibility. A comprehensive QC protocol should include:

• Identity verification through:

  • Mass spectrometry (exact mass determination with ±0.1 Da accuracy)

  • N-terminal sequencing (minimum 5-10 residues)

  • Amino acid composition analysis

• Purity assessment via:

  • Analytical RP-HPLC (minimum 95% purity)

  • Capillary electrophoresis

  • SDS-PAGE with silver staining

• Structural integrity confirmation through:

  • Circular dichroism spectroscopy

  • Disulfide bond mapping for cysteine-containing peptides

• Biological activity validation:

  • Receptor binding assays (affinity within 2-fold of reference standard)

  • Functional assays (EC50 within 3-fold of reference standard)

• Contaminant screening:

  • Endotoxin testing (<1 EU/mg peptide)

  • Host cell protein analysis (<100 ppm)

Acceptance criteria should be established for each parameter, and a certificate of analysis should accompany each batch, documenting lot-to-lot consistency. A reference standard should be maintained for comparative analysis. This methodological approach ensures research reproducibility and facilitates meaningful comparison of results across different studies and laboratories, similar to the verification approaches used for other recombinant proteins .