Recombinant Rana sevosa Ranatuerin-2SEc

What is the general structure of Ranatuerin-2SEc and how does it compare to other ranatuerin-2 peptides?

Ranatuerin-2SEc belongs to the ranatuerin-2 family, which typically features an N-terminal α-helix domain and a characteristic C-terminal "Rana box" with a disulfide bridge. The primary structures of ranatuerin-2 peptides are poorly conserved, except for several invariant features including Gly1 residue position and the conserved cysteine residues that form the cyclic hexapeptide or heptapeptide domain at the C-terminus . While specific sequence information for Ranatuerin-2SEc varies from other variants like ranatuerin-2-AW (R2AW) from Amolops wuyiensis (GFMDTAKNVAKNVAATLLDKLKCKITGGC), the general structural principles remain consistent across the family . The peptide would be expected to adopt an amphipathic helical structure with a hydrophobic face that interacts with bacterial membranes.

What analytical techniques are most appropriate for confirming the structural integrity of recombinant Ranatuerin-2SEc?

For structural characterization of recombinant Ranatuerin-2SEc, researchers should employ:

• Mass Spectrometry (MS): For confirming molecular weight and sequence

• Circular Dichroism (CD): To verify secondary structure elements (α-helical content)

• Nuclear Magnetic Resonance (NMR): For detailed three-dimensional structure analysis

• Disulfide Bond Analysis: Using reduction/alkylation followed by MS to confirm correct disulfide formation in the Rana box

These techniques should be used in combination to ensure proper folding and disulfide bond formation, which appears crucial for some biological functions, though studies on R2AW suggest the disulfide bridge may be dispensable for antibacterial activity in some ranatuerin-2 peptides .

What are the optimal expression systems for producing recombinant Ranatuerin-2SEc?

Based on studies with related peptides, the following expression systems are recommended:

• E. coli Expression Systems: Most commonly used for AMPs, typically employing fusion partners like thioredoxin, SUMO, or GST to reduce toxicity to the host and enhance solubility. The pET expression system with BL21(DE3) cells often provides good yields.

• Yeast Expression Systems: Pichia pastoris can be advantageous for disulfide-containing peptides like Ranatuerin-2SEc as it provides a eukaryotic environment with better folding machinery.

Expression protocols typically require optimization of induction conditions (temperature, IPTG concentration, induction time) to maximize yield while maintaining proper folding. For Ranatuerin-2SEc specifically, ensuring proper formation of the disulfide bridge in the Rana box domain requires careful consideration of the expression system and downstream processing.

What purification strategy yields the highest purity and recovery of bioactive Ranatuerin-2SEc?

A multi-step purification protocol is recommended:

• Initial Capture: Affinity chromatography using an appropriate tag (His-tag, GST)

• Tag Removal: Enzymatic cleavage using specific proteases (TEV, thrombin, Factor Xa)

• Polish Purification: Reversed-phase HPLC to separate the peptide from contaminants

• Oxidative Folding: Controlled oxidation in a glutathione redox buffer (GSH/GSSG) to ensure proper disulfide bond formation

This strategy typically yields 5-10 mg of pure peptide per liter of culture. Purification should be monitored by SDS-PAGE, mass spectrometry, and activity assays to ensure the recombinant peptide maintains structural integrity and bioactivity.

What is the spectrum of antimicrobial activity for Ranatuerin-2SEc compared to other family members?

Ranatuerin-2 family peptides generally show broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria . By comparing with characterized family members, Ranatuerin-2SEc would be expected to exhibit activity against pathogens including:

Studies on related peptide R2AW showed moderate antibacterial activity with MIC values of 32 μM against both S. aureus and E. coli . Ranatuerin-2PLx demonstrated less activity against MRSA with an MIC of 256 μM . Variations in antimicrobial potency across different strains highlight the importance of testing Ranatuerin-2SEc against a comprehensive panel of clinically relevant bacteria.

What is the primary mechanism of action for Ranatuerin-2SEc's antimicrobial activity?

Like other ranatuerin-2 peptides, Ranatuerin-2SEc likely acts primarily through membrane disruption. Research on optimized variants of R2AW demonstrated that the peptides kill bacteria through membrane permeabilization at a highly efficient rate . The mechanism typically involves:

• Initial electrostatic interaction between the cationic peptide and negatively charged bacterial membranes

• Insertion of hydrophobic regions into the membrane

• Disruption of membrane integrity through pore formation or carpet-like mechanisms

This rapid membrane-disruptive action makes the development of resistance less likely compared to conventional antibiotics with specific molecular targets. For experimental verification, researchers should employ membrane permeabilization assays (using fluorescent dyes like propidium iodide or SYTOX Green) and transmission electron microscopy to directly visualize membrane damage.

How does the Rana box domain contribute to the biological activity of Ranatuerin-2SEc?

The role of the Rana box (cyclic domain) in ranatuerin-2 peptides has been a subject of investigation. Studies on R2AW revealed interesting findings:

• Serine-substitution products ([Ser23,29]R2AW) where cysteines were replaced with serines (preventing disulfide formation) showed similar antibacterial activity to the natural peptide

• Cyclic-domain-deletion products (R2AW(1-22)) with complete removal of the Rana box also maintained antibacterial activity

• Native peptide with intact Rana box

• Linear variants with disrupted disulfide bonds

• Truncated variants lacking the entire C-terminal domain

Such comparative analysis would establish whether the dispensability of the Rana box is a general feature of the ranatuerin-2 family or specific to certain members.

What amino acid modifications have proven most effective in enhancing the biological activities of ranatuerin-2 peptides?

Studies on R2AW provide valuable insights into effective modification strategies:

• Enhanced Cationicity: Substituting neutral residues with lysine at positions 4 and 19 improved antimicrobial activity

• Increased Hydrophobicity: Replacing residues with more hydrophobic amino acids (e.g., leucine at position 20) enhanced membrane interaction

• C-terminal Amidation: Adding an amide group to the C-terminus improved antimicrobial activity and stability while potentially reducing cytotoxicity

The most successful variant created through these modifications was [Lys4,19, Leu20]R2AW(1-22)-NH2, which "exhibited significantly optimised antibacterial and anticancer activities" . This peptide showed dramatically improved MIC values:

This optimized variant demonstrates the potential for rational design of Ranatuerin-2SEc derivatives with enhanced therapeutic properties.

What evidence exists for anticancer activity of ranatuerin-2 peptides and what might this suggest for Ranatuerin-2SEc?

Several ranatuerin-2 family members have demonstrated anticancer properties:

• Ranatuerin-2PLx was found to inhibit the proliferation of several tumor cell lines, showing particular potency against prostate cancer cells (PC-3)

• The optimized variant [Lys4,19, Leu20]R2AW(1-22)-NH2 exhibited enhanced anticancer activities compared to the natural peptide

These findings suggest that Ranatuerin-2SEc may also possess anticancer potential. Studies with ranatuerin-2PLx demonstrated that the peptide induced early cell apoptosis within 6 hours of treatment, as observed by Annexin V-FITC/propidium iodide staining, along with activation of Caspase-3 at 5 μM peptide concentration .

What experimental approaches should be used to evaluate the anticancer potential of Ranatuerin-2SEc?

To thoroughly characterize the anticancer properties of Ranatuerin-2SEc, researchers should employ:

• Cell Viability Assays: MTT or MTS assays across multiple cancer cell lines to determine IC50 values

• Apoptosis Detection: Annexin V/PI staining followed by flow cytometry

• Caspase Activation Assays: Measuring activation of Caspase-3 and other apoptotic markers

• Cell Cycle Analysis: Flow cytometry with propidium iodide staining

• Mechanistic Studies: Western blot analysis of key signaling proteins involved in apoptosis

For comprehensive evaluation, testing should include a diverse panel of cancer cell lines (prostate, breast, lung, colorectal) alongside non-cancerous cell lines to assess selectivity. Based on findings with other ranatuerins, particular attention should be paid to prostate cancer cell lines like PC-3, where ranatuerin-2PLx showed pronounced effects .

How effective is Ranatuerin-2SEc against antibiotic-resistant bacterial strains?

While specific data for Ranatuerin-2SEc is limited, insights can be drawn from related peptides:

• The optimized variant [Lys4,19, Leu20]R2AW(1-22)-NH2 showed significant activity against MRSA with an MIC of 2 μM

• This variant also "exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model"

These findings suggest that properly designed ranatuerin-2 peptides can be effective against antibiotic-resistant bacteria. For Ranatuerin-2SEc, researchers should conduct systematic testing against a panel of clinically relevant resistant strains, including:

• Methicillin-resistant Staphylococcus aureus (MRSA)

• Vancomycin-resistant Enterococcus (VRE)

• Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

• Carbapenem-resistant Enterobacteriaceae (CRE)

• Multidrug-resistant Pseudomonas aeruginosa

What synergistic effects might be expected when combining Ranatuerin-2SEc with conventional antibiotics?

Antimicrobial peptides often show synergistic effects with conventional antibiotics through several mechanisms:

• Enhanced penetration of antibiotics across damaged bacterial membranes

• Simultaneous targeting of different bacterial components

• Reduction in the concentration of each agent required for efficacy

For Ranatuerin-2SEc, researchers should perform checkerboard assays with various classes of antibiotics to calculate fractional inhibitory concentration (FIC) indices. Particular attention should be paid to combinations with:

• β-lactams (for Gram-positive bacteria)

• Aminoglycosides (for Gram-negative bacteria)

• Glycopeptides (for resistant Gram-positive bacteria)

Such combination approaches may help reduce the required concentrations of both the peptide and conventional antibiotics, potentially minimizing toxicity while enhancing antimicrobial efficacy.

What in vivo models are most appropriate for evaluating Ranatuerin-2SEc efficacy?

Based on studies with related peptides, the following in vivo models are recommended:

• Waxworm (Galleria mellonella) Model: The optimized variant [Lys4,19, Leu20]R2AW(1-22)-NH2 showed efficacy in an MRSA-infected waxworm model . This model offers a cost-effective initial screen for in vivo activity.

• Mouse Infection Models: For more advanced testing, mouse models of specific infections should be employed:

  • Systemic infection models

  • Skin infection models

  • Pulmonary infection models

• Tumor Xenograft Models: For anticancer evaluation, tumor xenograft models using cell lines where the peptide shows significant in vitro activity

These models should assess not only efficacy but also pharmacokinetics, biodistribution, and potential toxicity of the peptide.

What are the critical considerations for advancing Ranatuerin-2SEc toward therapeutic development?

Several key factors must be addressed:

• Stability Optimization: Strategies to enhance proteolytic stability include:

  • D-amino acid substitutions

  • Terminal modifications (amidation, acetylation)

  • Cyclization beyond the natural disulfide bridge

• Toxicity Evaluation: Comprehensive testing should include:

  • Hemolysis assays

  • Cytotoxicity against normal human cell lines

  • Acute toxicity in animal models

• Formulation Development: Suitable delivery systems must be developed, such as:

  • Lipid-based nanoparticles

  • Polymer-based delivery systems

  • Controlled-release formulations

The promising candidate [Lys4,19, Leu20]R2AW(1-22)-NH2 demonstrates that rational design can significantly enhance the therapeutic potential of ranatuerin-2 peptides . Similar approaches could be applied to Ranatuerin-2SEc to develop an "appealing candidate for therapeutic development."