Recombinant Rana boylii Ranatuerin-2BYb

What is the primary structure of Ranatuerin-2BYb from Rana boylii?

Ranatuerin-2BYb belongs to the ranatuerin-2 family of peptides, which are characterized by a C-terminal cyclic hexapeptide domain rather than the more common heptapeptide found in other AMPs. The primary structure of ranatuerins has been poorly conserved across species, with several residue deletions and only five amino acids typically being invariant: Gly1, Ala15, Lys22, Cys23, and Cys28 . The two invariant cysteines form the cyclic domain at the C-terminus, which is a distinctive feature of this peptide family. While specific sequences vary between different frog species, the structural organization remains consistent with other ranatuerins found in North American ranid frogs.

How does the structure of Ranatuerin-2BYb compare to other ranatuerins?

Ranatuerin-2 peptides from Rana boylii share phylogenetic relationships with ranatuerins from R. luteventris, as they segregate within the same clade . This suggests evolutionary conservation of certain structural features. The predicted conformation of ranatuerins typically comprises three structural domains: an α-helix (residues 1-8), β-sheet (residues 11-16), and β-turn (residues 20-25) . This structural arrangement is critical for their antimicrobial function, with modifications to these regions often resulting in altered activity profiles.

What are the typical antimicrobial properties of Ranatuerin-2BYb?

Based on studies of related ranatuerins, these peptides exhibit broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The antimicrobial potency varies significantly among different ranatuerins, with reported MIC values against E. coli ranging from 2 μM to 30 μM, and against S. aureus from 2 μM to >200 μM . Activity against C. albicans is generally lower. The hemolytic activities of ranatuerin-2 peptides against human erythrocytes also vary, which is an important consideration for therapeutic potential.

What expression systems are most effective for recombinant production of Ranatuerin-2BYb?

• The antimicrobial nature of the peptide may be toxic to the host cells

• Proper formation of disulfide bridges is essential for bioactivity

• Proteolytic degradation may occur during expression

A recommended approach is to express the peptide as a fusion protein with a carrier protein (such as thioredoxin or SUMO) to neutralize toxicity and increase solubility. The fusion construct should include a specific protease cleavage site for subsequent release of the native peptide. For proper disulfide bridge formation, expression in the oxidizing environment of the periplasmic space or post-purification oxidative folding may be necessary.

What purification strategies optimize yield and maintain activity of recombinant Ranatuerin-2BYb?

A multi-step purification strategy is typically required:

• Initial capture using affinity chromatography targeting the fusion tag

• Enzymatic cleavage to release the peptide from the fusion partner

• Reverse-phase HPLC for final purification and removal of contaminants

• Quality control using mass spectrometry to confirm proper molecular weight and disulfide bridge formation

The purification protocol should minimize exposure to reducing agents that could disrupt the cyclic domain formed by the disulfide bridge. The pH of purification buffers should be carefully controlled, as many AMPs aggregate at their isoelectric point. Typical yields for recombinant AMPs range from 1-5 mg/L of bacterial culture, with purity exceeding 95% necessary for reliable bioactivity assays.

How does the cyclic domain (Rana box) of Ranatuerin-2BYb contribute to its antimicrobial activity?

In studies of ranatuerin-2-AW (R2AW), researchers found that both serine-substituted ([Ser23,29]R2AW) and cyclic-domain-deleted but C-terminally amidated (R2AW(1-22)-NH2) variants maintained similar antibacterial activity to the natural peptide . This suggests that for some ranatuerins, the disulfide bridge and Rana box may be dispensable for antibacterial activity, particularly when compensatory modifications like C-terminal amidation are present.

What residue modifications can enhance the therapeutic potential of Ranatuerin-2BYb?

Based on studies of ranatuerin analogues, several modification strategies have proven effective:

• Increasing cationicity by lysine substitutions at strategic positions

• Enhancing hydrophobicity through leucine substitutions

• C-terminal amidation to improve stability and antimicrobial potency

• Tryptophan substitutions to enhance membrane interactions

A particularly successful example is [Lys4,19, Leu20]R2AW(1-22)-NH2, which exhibited significantly enhanced antibacterial and anticancer activities compared to the native peptide . This variant demonstrated improved antimicrobial potency against both standard strains and multidrug-resistant bacteria, including MRSA.

How do structural modifications affect the hemolytic activity of Ranatuerin-2BYb?

The goal of rational design should be to maximize antimicrobial potency while minimizing hemolytic activity, which requires careful consideration of the amphipathicity and charge distribution of the modified peptide.

What are the optimal assays for evaluating antimicrobial activity of Ranatuerin-2BYb against drug-resistant bacteria?

Standard protocols for evaluating antimicrobial activity include:

• Minimum Inhibitory Concentration (MIC) Determination:

  • Broth microdilution method following CLSI guidelines

  • Testing against both reference strains and clinical isolates of multidrug-resistant bacteria

  • Important to test against MRSA, VRE, carbapenem-resistant Enterobacteriaceae, and Pseudomonas aeruginosa

• Minimum Bactericidal Concentration (MBC) Determination:

  • Subculturing from MIC plates onto antibiotic-free media

  • MBC is defined as the lowest concentration that reduces the viability of the initial bacterial inoculum by ≥99.9%

• Time-Kill Kinetics:

  • Monitoring bacterial viability at different time points (0, 0.5, 1, 2, 4, 8, and 24 h)

  • Provides information on the rate of bactericidal activity

  • Critical for understanding the peptide's mode of action

Data from studies on related ranatuerins show that peptides like [Lys4,19, Leu20]R2AW(1-22)-NH2 can achieve MIC values as low as 2-4 μM against MRSA and other resistant pathogens .

How can researchers effectively evaluate the membrane permeabilization mechanism of Ranatuerin-2BYb?

Membrane permeabilization is a common mechanism of action for AMPs like ranatuerins. The following methods provide comprehensive assessment:

• Fluorescent Dye Leakage Assays:

  • Using calcein or SYTOX Green to monitor membrane integrity

  • Propidium iodide uptake for assessing bacterial membrane damage

  • ONPG hydrolysis assay for outer membrane permeabilization in Gram-negative bacteria

• Membrane Depolarization Assays:

  • DiSC3(5) or DiBAC4(3) dyes to monitor changes in membrane potential

  • Rapid depolarization typically indicates pore formation

• Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM):

  • Direct visualization of membrane damage and morphological changes

  • Can reveal specific mechanisms (e.g., carpet model vs. barrel-stave model)

Studies of ranatuerin analogues like RPb have shown rapid bacterial killing via membrane permeabilization without damaging erythrocyte cell membranes , indicating selective toxicity toward bacterial membranes.

What animal models are most appropriate for evaluating the in vivo efficacy of Ranatuerin-2BYb?

Based on successful studies with related ranatuerins, the following models are recommended:

• Galleria mellonella (Greater Wax Moth) Larval Model:

  • Economical and ethically advantageous invertebrate model

  • Allows for high-throughput screening

  • Suitable for preliminary efficacy and toxicity studies

  • Studies have shown that ranatuerin analogues like RPb and [Lys4,19, Leu20]R2AW(1-22)-NH2 significantly decreased mortality in S. aureus-infected G. mellonella larvae

• Murine Infection Models:

  • Systemic infection models (intraperitoneal or intravenous)

  • Localized infection models (skin, soft tissue, urinary tract)

  • More relevant for assessing pharmacokinetics and immunomodulatory effects

• Ex Vivo Human Skin Models:

  • For testing topical applications

  • Provides information on penetration and local toxicity

The G. mellonella model has become increasingly popular for preliminary in vivo testing due to its cost-effectiveness and correlation with mammalian models. In studies with [Lys4,19, Leu20]R2AW(1-22)-NH2, treatment significantly improved survival rates in MRSA-infected waxworm models .

How should researchers address potential immunogenicity concerns with Ranatuerin-2BYb?

Peptide therapeutics may induce immune responses that can limit efficacy or cause adverse reactions. To address immunogenicity:

• In Silico Prediction:

  • Use immunoinformatics tools to identify potential T-cell and B-cell epitopes

  • Modify sequences to reduce immunogenic regions while preserving activity

• In Vitro Assays:

  • Human peripheral blood mononuclear cell (PBMC) proliferation assays

  • Cytokine release assays to detect pro-inflammatory responses

  • Complement activation studies

• Formulation Strategies:

  • PEGylation or other chemical modifications to reduce immunogenicity

  • Encapsulation in liposomes or nanoparticles to shield from immune recognition

• Analysis of Neutralizing Antibodies:

  • Development of assays to detect antibodies that could neutralize peptide activity

  • Monitoring of repeated dose studies for efficacy reduction

How does Ranatuerin-2BYb interact with conventional antibiotics in combination therapy?

Antimicrobial peptides like ranatuerins often show synergistic effects with conventional antibiotics. Researchers should evaluate:

• Checkerboard Assays:

  • Determine fractional inhibitory concentration index (FICI)

  • FICI ≤0.5 indicates synergy, 0.5-1.0 indicates additivity, >1.0-4.0 indicates indifference, >4.0 indicates antagonism

• Time-Kill Curve Analysis:

  • Assess if combinations result in faster or more complete killing than either agent alone

  • Particularly valuable for determining the kinetics of synergistic interactions

• Post-Antibiotic Effect (PAE) Studies:

  • Evaluate if combinations extend the duration of antimicrobial effects after removal of the agents

• Mechanistic Studies:

  • Investigate whether AMPs increase antibiotic penetration via membrane permeabilization

  • Assess if combinations prevent resistance development through different targeting mechanisms

Combinations should be tested against both susceptible and resistant bacterial strains to identify potentially valuable therapeutic approaches for difficult-to-treat infections.

Can Ranatuerin-2BYb be effectively formulated to overcome stability and delivery challenges?

AMPs face several challenges related to stability and delivery. Research should focus on:

• Protease Resistance:

  • D-amino acid substitutions at susceptible positions

  • Terminal modifications (acetylation, amidation) to protect from exopeptidases

  • Cyclization strategies beyond the native disulfide bridge

• Delivery Systems:

  • Liposomal formulations to protect from proteolytic degradation

  • Polymer-based nanoparticles for controlled release

  • Hydrogels for topical or wound healing applications

• Stability Testing:

  • Accelerated stability studies under various pH and temperature conditions

  • Serum stability assays to predict in vivo half-life

  • Long-term storage stability in different formulations

• Administration Routes:

  • Evaluation of bioavailability via different routes (topical, intranasal, inhalation, oral with absorption enhancers)

  • Assessment of tissue distribution and pharmacokinetics

How might genomic and transcriptomic approaches enhance our understanding of Ranatuerin-2BYb evolution and function?

Advanced research approaches include:

• Comparative Genomics:

  • Sequence analysis across Rana species to trace evolutionary patterns

  • Identification of selection pressures on ranatuerin genes

  • Analysis of gene duplication events that have given rise to peptide diversity

• Transcriptome Analysis:

  • RNA-seq of frog skin tissue to understand expression patterns

  • Comparison of expression levels under different environmental stressors

  • Identification of regulatory elements controlling expression

• Population Genetics:

  • Studying polymorphisms in ranatuerin genes across populations

  • Correlating peptide variants with habitat and pathogen exposure

  • Investigating how anthropogenic factors affect AMP diversity

Phylogenetic analysis has already demonstrated that ranatuerins from R. boylii and R. luteventris segregate within the same clade , providing insight into their evolutionary history and potential functional similarities.

What are the emerging applications of Ranatuerin-2BYb beyond antimicrobial therapy?

Research on ranatuerins has revealed potential applications beyond traditional antimicrobial therapy:

• Anti-Biofilm Activity:

  • Studies show that ranatuerins can both prevent biofilm formation and eradicate established biofilms

  • Particularly relevant for device-associated and chronic infections

  • Modified ranatuerins like [Lys4,19, Leu20]R2AW(1-22)-NH2 show potent activity against biofilm formation with MBIC values ranging from 4 μM to 16 μM

• Anticancer Properties:

  • Some ranatuerins have demonstrated selective cytotoxicity against cancer cells

  • Potential for combination with conventional chemotherapy

  • Mechanism likely involves membrane disruption and possibly apoptosis induction

  • Peptides from the ranatuerin-2 family like ranatuerin-2PLx can prevent cancer cell proliferation

• Immunomodulatory Effects:

  • AMPs can modulate immune responses independently of direct antimicrobial action

  • Potential applications in inflammatory and autoimmune conditions

  • Investigation of effects on cytokine production and immune cell activation

• Wound Healing Applications:

  • Combination of antimicrobial, anti-biofilm, and potential pro-healing properties

  • Development of biomaterial-peptide composites for advanced wound dressings

How can computational approaches accelerate the design of optimized Ranatuerin-2BYb derivatives?

Computational methods offer powerful tools for peptide optimization:

• Molecular Dynamics Simulations:

  • Predicting conformational changes in different environments

  • Understanding membrane interactions at atomic resolution

  • Identifying key residues for activity

• Machine Learning-Based Design:

  • Training algorithms on structure-activity data from multiple AMPs

  • Predicting optimal amino acid substitutions for desired properties

  • Balancing antimicrobial potency with reduced toxicity

• Quantitative Structure-Activity Relationship (QSAR) Models:

  • Developing models to relate physicochemical properties to biological activities

  • Identifying patterns not obvious from conventional analysis

  • Guiding rational design of next-generation derivatives

• Virtual Screening:

  • In silico testing of large libraries of potential derivatives

  • Prioritizing candidates for experimental validation

  • Accelerating the discovery process while reducing resource requirements

The rational design approach demonstrated with R2AW, where progressive modifications significantly enhanced antimicrobial and anticancer activities , provides a framework that can be further optimized through computational methods.