Recombinant Rana sevosa Ranatuerin-2SEb

What is Ranatuerin-2SEb and what is its biological origin?

Ranatuerin-2SEb is one of seven novel peptides isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa (also known as Lithobates sevosus). It belongs to the ranatuerin-2 family of antimicrobial peptides . The peptide was named following the accepted terminology for ranid frog antimicrobial peptides, with "ranatuerin" indicating the peptide family, "2" denoting the specific subfamily, "SE" representing the species of origin (sevosa), and "b" indicating the specific isotype .

What are the primary structural characteristics of Ranatuerin-2SEb?

Ranatuerin-2SEb, like other members of the ranatuerin-2 family, likely contains a C-terminal cyclic domain formed by a disulfide bridge between two cysteine residues, commonly known as the "Rana box" . Based on studies of similar ranatuerins, it probably exhibits an α-helical structure in membrane-mimicking environments while adopting a random coil conformation in aqueous solutions . The peptide is expected to have cationic properties and amphipathic structure, which are typical characteristics of antimicrobial peptides that facilitate interaction with negatively charged bacterial membranes.

What biological activities have been documented for Ranatuerin-2SEb?

Ranatuerin-2SEb, similar to other peptides isolated from Rana sevosa skin secretions, demonstrates both antimicrobial activity and histamine-releasing properties . This dual functionality is particularly interesting from an evolutionary perspective, as it suggests these peptides may serve multiple defensive roles in the frog's innate immune system. The peptide likely exhibits broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria, and possibly fungi, similar to other ranatuerins .

What expression systems are most suitable for recombinant Ranatuerin-2SEb production?

For recombinant expression of Ranatuerin-2SEb, E. coli-based systems are commonly employed for antimicrobial peptides. Based on methodologies used for similar peptides, a strategy involving fusion protein expression is recommended to overcome potential toxicity to the host cells. The peptide gene can be cloned following techniques similar to the "shotgun" cloning method described for Ranatuerin-2Pb, where specific primers are used in 3'-RACE reactions . Expression as a fusion with partners such as thioredoxin, SUMO, or MBP can enhance solubility and reduce toxicity to the expression host.

What are the critical considerations for designing a recombinant Ranatuerin-2SEb construct?

When designing a recombinant construct, several factors must be considered:

• Codon optimization for the expression host

• Inclusion of appropriate protease cleavage sites for fusion tag removal

• Strategy for disulfide bond formation (if preserving the Rana box)

• N-terminal and C-terminal modifications that might affect activity

Studies on related ranatuerins suggest that C-terminal amidation might enhance antimicrobial activity, as seen with RPb, a truncated analogue of Ranatuerin-2Pb . Additionally, consideration should be given to whether the cyclic domain (Rana box) is necessary for activity, as some studies indicate it may be dispensable for certain antimicrobial functions .

What purification strategies yield the highest purity and activity for recombinant Ranatuerin-2SEb?

A multi-step purification strategy is typically required:

• Initial capture using affinity chromatography based on the fusion tag

• Proteolytic cleavage to remove the fusion partner

• Reverse-phase HPLC for final purification

• Controlled oxidation conditions for proper disulfide bond formation

For the synthesis and purification of similar ranatuerins, solid-phase synthesis methods followed by air-oxidation at room temperature for three days have been employed to form the disulfide bond . Purification to >95% purity can be achieved using RP-HPLC, with confirmation of molecular mass by electrospray mass spectrometry or MALDI-TOF mass spectrometry .

How can the secondary structure of Ranatuerin-2SEb be accurately characterized?

Circular dichroism (CD) spectroscopy is the primary method for analyzing the secondary structure of ranatuerins in different environments. Based on studies of related peptides, the secondary structure should be analyzed in:

• Helical propagating solution (50% TFE/H₂O)

• Aqueous solution (H₂O)

• Lipid bilayer liposomes mimicking bacterial membranes (POPC/POPG 1:1 for S. aureus and POPE/POPG 3:1 for E. coli)

The estimated secondary structure contents from CD analysis of related ranatuerins in different environments provide a reference for comparison:

This table illustrates how the helical content significantly increases in membrane-mimicking environments compared to aqueous solution .

What role does the "Rana box" play in the structure and function of Ranatuerin-2SEb?

The functional significance of the "Rana box" (C-terminal cyclic domain) appears to vary among different ranatuerins. In studies of Ranatuerin-2-AW from Amolops wuyiensis, researchers found that serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide, suggesting that the disulfide bridge and Rana box were dispensable for antibacterial activity .

How do environmental factors affect the structural dynamics of Ranatuerin-2SEb?

Environmental factors significantly influence the structural dynamics of ranatuerins. CD spectroscopy studies of related peptides show that:

• In aqueous environments, ranatuerins predominantly exhibit random coil structures

• In membrane-mimicking environments (TFE/H₂O mixtures or lipid bilayers), they form substantial α-helical structures

• The degree of α-helical formation may differ between mammalian and bacterial membrane mimics

This conformational flexibility is likely crucial for the peptide's function, allowing it to adopt an active conformation upon interaction with bacterial membranes. NMR spectroscopy in different environments would provide more detailed insights into the three-dimensional structural changes of Ranatuerin-2SEb under varying conditions.

What methods are most reliable for assessing the antimicrobial activity of Ranatuerin-2SEb?

Several complementary methods should be employed for robust characterization of antimicrobial activity:

• Minimum Inhibitory Concentration (MIC) assays using broth microdilution methods against a panel of Gram-positive bacteria, Gram-negative bacteria, and fungi

• Minimum Bactericidal Concentration (MBC) determination

• Time-kill kinetics to assess the rate of bactericidal activity

• Biofilm inhibition and eradication assays, as some ranatuerins have demonstrated anti-biofilm properties

• Membrane permeabilization assays to investigate the mechanism of action

These assays should be performed using standardized protocols, such as those recommended by the Clinical and Laboratory Standards Institute (CLSI), with appropriate positive controls (conventional antibiotics or well-characterized antimicrobial peptides) and negative controls.

How does Ranatuerin-2SEb's antimicrobial mechanism of action compare to other ranatuerins?

Based on studies of related ranatuerins, the mechanism of action likely involves membrane permeabilization. For example, RPb (a truncated analogue of Ranatuerin-2Pb) was shown to kill bacteria rapidly via membrane permeabilization . Specifically designed experiments to elucidate Ranatuerin-2SEb's mechanism would include:

• Membrane permeabilization assays using fluorescent dyes

• Liposome leakage assays with model membranes of different compositions

• Electron microscopy to visualize membrane damage

• Electrophysiology techniques to detect pore formation

• Studies with fluorescently labeled peptide to track localization

These experiments would determine whether Ranatuerin-2SEb acts primarily through membrane disruption or if it has additional intracellular targets.

What structural modifications could enhance the antimicrobial activity of Ranatuerin-2SEb while minimizing cytotoxicity?

Strategic modifications that might enhance antimicrobial activity based on studies of related peptides include:

• C-terminal amidation, which enhanced activity in RPb (a truncated analogue of Ranatuerin-2Pb)

• Targeted substitutions to increase cationicity and optimize hydrophobicity, as demonstrated for [Lys⁴,¹⁹, Leu²⁰]R2AW(1-22)-NH₂, which exhibited significantly optimized antibacterial and anticancer activities

• Truncation to eliminate non-essential regions while maintaining the active core

To minimize cytotoxicity, modifications should aim to enhance selectivity for bacterial over mammalian membranes, potentially by:

What in vivo models are most appropriate for evaluating Ranatuerin-2SEb efficacy against bacterial infections?

Based on studies of related ranatuerins, both invertebrate and vertebrate models can be employed:

• Galleria mellonella (waxworm) infection model: This has been successfully used to evaluate the in vivo efficacy of RPb against S. aureus infections, demonstrating reduced mortality in infected larvae

• Murine infection models: These would include systemic infection models, skin infection models, or pulmonary infection models depending on the target pathogens

• Ex vivo tissue models: These can bridge the gap between in vitro and in vivo testing

The waxworm model offers advantages of ethical and practical simplicity while still providing valuable data on in vivo efficacy. For RPb, researchers demonstrated that it decreased the mortality of S. aureus-infected Galleria mellonella, suggesting this model could be appropriate for initial in vivo testing of Ranatuerin-2SEb .

How can synergistic interactions between Ranatuerin-2SEb and conventional antibiotics be identified and optimized?

Synergistic interactions can be systematically evaluated using:

• Checkerboard assays to determine Fractional Inhibitory Concentration (FIC) indices

• Time-kill synergy assays to assess the rate and extent of bacterial killing

• Post-antibiotic effect studies to evaluate potential enhancement

• Mechanistic studies to understand the basis of synergy (e.g., increased membrane permeabilization allowing better access of antibiotics to intracellular targets)

Optimization strategies would include:

• Identifying the most synergistic antibiotic classes

• Determining optimal concentration ratios

• Exploring sequential versus simultaneous administration

• Investigating formulation approaches to enhance co-delivery

What approaches can determine the potential of Ranatuerin-2SEb against antibiotic-resistant pathogens?

To evaluate efficacy against antibiotic-resistant pathogens:

• Activity testing against clinical isolates with defined resistance mechanisms, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multidrug-resistant Gram-negative bacteria

• Assessment of cross-resistance by generating peptide-resistant mutants and testing susceptibility to conventional antibiotics

• Evaluation of activity in biofilm models, as biofilms often contribute to antibiotic resistance

• Investigation of resistance development through serial passage experiments

• Genomic and transcriptomic analyses to identify resistance mechanisms if they emerge

Studies on related ranatuerins have shown activity against MRSA , suggesting Ranatuerin-2SEb may also be effective against this important resistant pathogen.

What are the most predictive cytotoxicity assays for evaluating Ranatuerin-2SEb safety?

A comprehensive cytotoxicity assessment would include:

• Hemolysis assays on erythrocytes to evaluate membrane-disruptive effects on mammalian cells

• MTT or similar viability assays on multiple human cell lines, including:

  • Lung epithelial cells (e.g., H157)

  • Breast cancer cells (e.g., MCF-7, MDA-MB-435s)

  • Neuronal cells (e.g., U251MG)

  • Prostate cancer cells (e.g., PC-3)

• LDH release assays to assess membrane integrity

• Specific toxicity assays for potential target organs (e.g., hepatocytes, renal cells)

For related ranatuerins, hemolysis assays have shown varying degrees of toxicity. For example, Ranatuerin-2Pb, RPa, and RPb exhibited hemolysis rates near 20% on horse erythrocytes at concentrations of 8, 32, and 64 μM, respectively, with HC₅₀ values of 16.11, 63.90, and 178.0 μM .

How can the therapeutic index of Ranatuerin-2SEb be calculated and optimized?

The therapeutic index (TI) is calculated as the ratio of the concentration causing toxicity to mammalian cells (typically HC₅₀ in hemolysis assays or IC₅₀ in cytotoxicity assays) to the concentration required for antimicrobial activity (MIC):

TI = HC₅₀ or IC₅₀ / MIC

Optimization strategies to improve the TI include:

• Structural modifications to reduce interactions with mammalian cell membranes while maintaining bacterial membrane activity

• Formulation approaches to enhance selective delivery to infection sites

• Conjugation with targeting moieties to increase specificity

• PEGylation or other modifications to reduce non-specific interactions

Studies on RPb (a truncated analogue of Ranatuerin-2Pb) showed it exhibited the highest therapeutic index among the peptides studied , suggesting that similar modifications might improve the TI of Ranatuerin-2SEb.

What stability studies are essential for developing Ranatuerin-2SEb as a therapeutic candidate?

Comprehensive stability assessment should include:

• Physical stability:

  • Temperature stability at various storage conditions

  • pH stability across physiological range

  • Freeze-thaw stability

• Chemical stability:

  • Oxidative stability (especially for methionine or cysteine residues)

  • Deamidation susceptibility

  • Aggregation propensity

• Biological stability:

  • Serum stability assays

  • Resistance to specific proteases (trypsin, chymotrypsin, etc.)

  • Tissue homogenate stability

• Formulation stability:

  • Compatibility with excipients

  • Long-term stability in final formulation

These studies would inform both storage conditions and potential modification strategies to enhance stability for therapeutic applications.

What analytical methods provide the most accurate characterization of recombinant Ranatuerin-2SEb?

A comprehensive analytical package would include:

• Primary structure confirmation:

  • Mass spectrometry (MS): Electrospray ionization MS and MALDI-TOF MS for molecular weight confirmation

  • Edman degradation for N-terminal sequencing

  • Amino acid analysis for composition verification

• Secondary and tertiary structure analysis:

  • Circular dichroism spectroscopy for secondary structure determination in different environments

  • NMR spectroscopy for three-dimensional structure determination

  • Fourier-transform infrared spectroscopy (FTIR) as a complementary technique for secondary structure

• Purity assessment:

  • Reverse-phase HPLC for purity determination (>95% purity is typically required)

  • Capillary electrophoresis as an orthogonal method

  • Size-exclusion chromatography to detect aggregates

• Functional characterization:

  • Antimicrobial activity assays against reference strains

  • Hemolysis assays for batch-to-batch consistency in safety profile

How can batch-to-batch consistency be ensured in recombinant Ranatuerin-2SEb production?

Ensuring batch-to-batch consistency requires:

• Establishment of well-defined Critical Quality Attributes (CQAs):

  • Molecular identity (mass, sequence)

  • Purity profile

  • Secondary structure content

  • Bioactivity (MIC against reference strains)

  • Cytotoxicity profile

• Implementation of robust analytical methods:

  • Validated HPLC methods for purity and identity

  • Standardized bioactivity assays with reference standards

  • Consistent CD spectroscopy protocols

• Process controls:

  • Defined cell banking system

  • Consistent fermentation parameters

  • Validated purification protocols

  • In-process testing at critical steps

• Statistical process control:

  • Trend analysis of quality attributes across batches

  • Establishment of acceptance criteria based on statistical analysis of historical data

What are the most sensitive methods for detecting host cell protein and DNA contamination in purified Ranatuerin-2SEb preparations?

For host cell protein (HCP) detection:

• ELISA using antibodies raised against the host cell proteome

• LC-MS/MS-based proteomic approaches for identification and quantification of specific HCPs

• Western blotting for detection of common HCP contaminants

For host cell DNA detection:

• Quantitative PCR (qPCR) targeting host-specific sequences

• Threshold assays using fluorescent DNA-binding dyes

• Next-generation sequencing for detailed characterization of DNA contaminants

Establishing acceptance criteria based on regulatory guidelines (typically <100 pg DNA per dose and <100 ppm HCP) is essential for advancing toward clinical applications.

How might structural biology techniques advance our understanding of Ranatuerin-2SEb's mechanism of action?

Advanced structural biology approaches could provide critical insights:

• Solution NMR studies in membrane-mimicking environments to determine the three-dimensional structure and orientation of Ranatuerin-2SEb when interacting with membranes

• Solid-state NMR to study peptide-membrane interactions in native-like bilayers

• Cryo-electron microscopy to visualize membrane perturbations or pore formation

• X-ray crystallography of the peptide in complex with potential protein targets

• Molecular dynamics simulations to model peptide-membrane interactions and conformational changes

These approaches would help elucidate whether Ranatuerin-2SEb acts primarily through carpet-like disruption, toroidal pore formation, barrel-stave pores, or other mechanisms.

What genomic approaches could identify novel Ranatuerin variants with enhanced properties?

Genomic and transcriptomic approaches to discover novel ranatuerins include:

• Next-generation sequencing of skin secretion transcriptomes from diverse ranid frog species

• Bioinformatic mining of existing amphibian genomic databases

• Comparative genomics to identify evolutionary patterns in antimicrobial peptide genes

• Molecular phylogenetic analysis to understand evolutionary relationships between different ranatuerins

These approaches could identify natural variants with enhanced properties or guide rational design of improved synthetic analogues. The "shotgun" cloning approach described for Ranatuerin-2Pb could serve as a starting methodology .

How might Ranatuerin-2SEb be developed for clinical applications beyond direct antimicrobial use?

Potential alternative applications include:

• Immunomodulatory applications, given the dual antimicrobial and histamine-releasing properties reported for peptides from Rana sevosa

• Anti-biofilm coatings for medical devices, based on the anti-biofilm properties demonstrated by some ranatuerins

• Combination therapy with conventional antibiotics to combat resistant infections

• Anticancer applications, as some ranatuerins have shown anticancer activity

• Templates for developing peptidomimetics with improved pharmacological properties

Exploring these diverse applications would require specialized in vitro and in vivo models tailored to each potential indication.