Recombinant Pelophylax ridibundus Urotensin-2 (UTS2)

What is Pelophylax ridibundus Urotensin-2 and what makes it scientifically relevant?

Pelophylax ridibundus Urotensin-2 is a peptide hormone belonging to the urotensin family, isolated from the marsh frog (Pelophylax ridibundus). This species is taxonomically significant as one of the parent species of the European edible frog (Pelophylax kl. esculentus), a hybrid between P. lessonae and P. ridibundus . UTS2 has gained research attention due to its potential pharmacological properties, similar to other bioactive peptides isolated from amphibian species. Amphibian-derived peptides often possess unique structural characteristics and bioactivities that can serve as templates for novel therapeutic compounds, particularly those with vasoactive, antimicrobial, or neuromodulatory activities.

Which expression systems are most appropriate for producing recombinant Pelophylax ridibundus UTS2?

Based on the available data for recombinant protein production from Pelophylax ridibundus, several expression systems have been successfully employed, each with distinct advantages depending on research requirements:

Selection should be based on specific research requirements, particularly whether native post-translational modifications are critical for the intended application.

How does recombinant UTS2 compare to naturally isolated UTS2?

While naturally isolated UTS2 from Pelophylax ridibundus might contain the authentic post-translational modifications, recombinant production offers several advantages including consistent purity, sequence verification, and the ability to incorporate specific modifications or tags. The bioactivity comparison between natural and recombinant UTS2 should be systematically evaluated through receptor binding assays, cell signaling studies, and functional tests. Similar bioactive peptides from amphibian species show variations in antimicrobial activities against different pathogens, as seen with peptides like ranatuerin-1, which exhibits activity against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis .

What are critical elements of robust experimental design when working with recombinant Pelophylax ridibundus UTS2?

Effective experimental design with recombinant UTS2 should follow structured methodological frameworks as outlined in experimental design literature. Researchers should:

• Clearly define research objectives and identify critical control points in the experimental process

• Select appropriate measurement techniques that accurately capture the biological responses of interest

• Implement proper randomization and controlled variable manipulation

• Account for restrictions on randomization using appropriate designs (e.g., randomized block or split-plot designs)

• Apply multivariate statistical techniques to analyze complex datasets generated from UTS2 experiments

As noted in experimental design methodology literature, "the main goal is to present a general framework for looking at an industrial experimental problem—starting from the problem definition stage, utilizing an appropriate experimental design, taking proper response measurements using techniques that describe the desired phenomenon that is to be studied and finally analyzing the data using multivariate techniques" .

How should dose-response experiments with UTS2 be designed?

For rigorous dose-response evaluations:

• Establish a logarithmic concentration range spanning at least 4-5 orders of magnitude

• Include appropriate positive controls (e.g., human UTS2) and negative controls (vehicle, unrelated peptides)

• Use factorial or fractional-factorial designs when evaluating UTS2 activity in combination with other factors

• Implement split-plot designs when randomization restrictions exist for certain experimental factors

• Ensure sufficient biological and technical replicates for statistical power

• Consider time-dependent effects by incorporating multiple measurement timepoints

The experimental design should account for practical implementation limitations, as "using seven different variables (two WP and five SP) in a full factorial setup, a 27 design would require 128 experiments. This is far too many for practical implementation" . Therefore, fractional factorial designs may be more appropriate.

What analytical techniques are most effective for characterizing recombinant Pelophylax ridibundus UTS2?

Comprehensive characterization should employ multiple complementary techniques:

• Mass spectrometry (MS) for molecular weight confirmation and sequence verification

• Circular dichroism (CD) spectroscopy for secondary structure analysis

• Near-Infrared spectroscopy (NIRS) for rapid assessment of structural properties, which "has been shown suitable not only as a rapid measurement technique for detecting changes in the final product quality at an early stage, but also for process control at the critical control point"

• High-performance liquid chromatography (HPLC) for purity determination

• Surface plasmon resonance (SPR) for binding kinetics analysis

• Nuclear magnetic resonance (NMR) for detailed structural characterization

• Bioactivity assays using appropriate cellular models expressing UTS2 receptors

What approaches can detect structural differences between Pelophylax ridibundus UTS2 and UTS2 from other species?

Comparative structural analysis should incorporate:

• Sequence alignment analysis to identify conserved and variable regions

• Predictive modeling based on known UTS2 structures from other species

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to compare solution conformations

• X-ray crystallography or NMR studies of receptor-bound states

• Molecular dynamics simulations to compare conformational flexibility

• Cross-species receptor binding studies to evaluate functional conservation

What purification strategy yields the highest purity recombinant Pelophylax ridibundus UTS2?

A multi-step purification strategy typically yields the best results:

• Initial capture using affinity chromatography (His-tag, GST-tag) depending on the expression construct

• Intermediate purification using ion-exchange chromatography

• Polishing step using size-exclusion chromatography

• Consider specialized techniques like biotinylation using AviTag-BirA technology, which "catalyzes amide linkage between the biotin and the specific lysine of the AviTag"

• Final validation using analytical techniques (MS, HPLC) to confirm purity and integrity

For E. coli-expressed UTS2, inclusion body extraction and refolding protocols may be necessary, followed by purification of correctly folded protein.

How can expression yields of recombinant Pelophylax ridibundus UTS2 be optimized in bacterial systems?

Optimization strategies include:

• Codon optimization based on E. coli codon usage preferences

• Selection of appropriate E. coli strains (BL21(DE3), Rosetta, etc.) based on rare codon content in the UTS2 sequence

• Optimization of induction parameters (temperature, IPTG concentration, induction timing)

• Co-expression with chaperones to improve folding

• Use of fusion partners (SUMO, thioredoxin, etc.) to enhance solubility

• Optimization of lysis and extraction protocols

• Implementation of auto-induction media systems for higher cell densities

How can recombinant Pelophylax ridibundus UTS2 be effectively biotinylated for detection or immobilization studies?

Site-specific biotinylation using AviTag technology offers significant advantages:

• Incorporate an AviTag sequence into the UTS2 construct during cloning

• Express the fusion protein in E. coli or other suitable expression systems

• Perform enzymatic biotinylation using E. coli biotin ligase (BirA), which "is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide"

• Purify the biotinylated product using standard chromatographic techniques

• Confirm biotinylation efficiency using streptavidin binding assays

The advantage of this approach is that "this recombinant protein was biotinylated in vivo by AviTag-BirA technology, which method is BirA catalyzes amide linkage between the biotin and the specific lysine of the AviTag" , ensuring specific and controlled biotinylation.

What approaches are recommended for structure-activity relationship studies with Pelophylax ridibundus UTS2?

Systematic structure-activity relationship studies should employ:

• Alanine scanning mutagenesis to identify critical residues for activity

• Truncation studies to define minimal active domains

• Chimeric constructs with UTS2 from other species to identify functionally conserved regions

• D-amino acid substitutions to enhance stability while maintaining activity

• Cyclization or constraint strategies to stabilize bioactive conformations

• Correlation of structural modifications with receptor binding affinities and downstream signaling pathways

For each variant, comprehensive characterization should include receptor binding assays, signaling pathway activation studies, and relevant functional assays.

How should researchers address inconsistent results in UTS2 activity assays?

When facing inconsistency in UTS2 activity data:

• Verify protein quality through analytical characterization (MS, HPLC)

• Examine storage conditions and avoid repeated freeze-thaw cycles

• Test for batch-to-batch variation in protein preparations

• Validate receptor expression levels in cell-based assays

• Control environmental variables (pH, temperature, buffer composition)

• Include appropriate positive controls in each experiment

• Consider time-dependent effects and receptor desensitization

Implementing factorial experimental designs can help identify interaction effects between variables that may contribute to inconsistent results, as these designs "are useful for developing and optimizing processes, and for the design of products with improved performance" .

How can researchers resolve contradictory findings between UTS2 expressed in different systems?

To reconcile contradictory results between expression systems:

• Conduct comprehensive comparative characterization of post-translational modifications

• Compare secondary and tertiary structures using spectroscopic techniques

• Assess aggregation state and oligomerization tendency

• Utilize multiple orthogonal activity assays to evaluate functional comparability

• Screen for host cell protein contaminants that might affect activity

• Standardize protein quantification methods across preparations

• Consider using multivariate analysis techniques to identify patterns in complex datasets

The source of expression can significantly impact protein properties as each system (E. coli, yeast, baculovirus, and mammalian cells) produces proteins with different post-translational modifications .

What experimental controls are essential when using recombinant Pelophylax ridibundus UTS2 in receptor binding studies?

Essential controls include:

• Positive control: Well-characterized UTS2 from human or other species with known binding properties

• Negative control: Structurally similar but non-binding peptide

• Competition control: Unlabeled UTS2 to demonstrate specific displacement

• Vehicle control: Buffer solution used for diluting UTS2

• Receptor expression control: Validation of receptor expression levels between experiments

• Non-specific binding control: Cells lacking the UTS2 receptor

• Data quality control: Saturation binding analysis to confirm binding site occupancy

How can chromosome engineering and marker-assisted techniques advance UTS2 research?

Emerging technologies in chromosome engineering and marker development could enhance UTS2 research:

• Development of molecular markers for UTS2 gene identification across amphibian species

• Application of CRISPR-Cas9 genome editing for introducing specific modifications to the UTS2 gene

• Implementation of chromosome engineering techniques similar to those used for "transfer of alien genes from wild relatives into modern crops"

• Development of UTS2-specific markers that are "tightly linked to [the gene] and provide useful resources" for tracking UTS2 variants in different Pelophylax species

• Application of "Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM)" for studying UTS2 gene expression and localization

These approaches parallel advancements in plant genetics where "chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops" , and could be adapted for amphibian peptide research.

What novel applications could emerge from comparative studies of UTS2 across different amphibian species?

Comparative studies of UTS2 across amphibian species could lead to:

• Discovery of novel UTS2 variants with enhanced stability or receptor selectivity

• Identification of conserved structural elements essential for activity

• Development of peptide-based therapeutic candidates

• Understanding evolutionary adaptation of UTS2 function across species

• Insights into species-specific physiological roles of UTS2

• Biomarker development for environmental monitoring

Similar to how "Ranatuerin-1 peptides were only isolated from three closely related species" , comparative studies could reveal unique UTS2 variants in Pelophylax species with potentially distinctive bioactivities.