Recombinant Rana japonica Temporin-1Ja

What is the molecular structure of Temporin-1Ja from Rana japonica?

Temporin-1Ja from Rana japonica is a 13-amino acid peptide with the sequence ILPLVGNLLNDLL.NH₂. The peptide has an amidated C-terminus, which is a common post-translational modification in amphibian antimicrobial peptides. Structurally, Temporin-1Ja belongs to the temporin family, which typically consists of short (10-14 amino acids), moderately hydrophobic peptides with limited cationic properties. Unlike some other antimicrobial peptides from Rana species, Temporin-1Ja does not contain cysteine residues and therefore lacks disulfide bonds, maintaining a linear conformation that adopts an α-helical structure in membrane-mimicking environments .

How does Temporin-1Ja differ structurally from other temporins in amphibian species?

Temporin-1Ja from Rana japonica shares the characteristic short sequence and α-helical structure common to temporin family peptides but demonstrates specific sequence variations. While structurally similar to temporins from other Rana species such as temporin-1DRa from Rana draytonii and temporin-1Va from Rana virgatipes, each possesses unique amino acid compositions affecting their antimicrobial properties. Unlike temporin-1CEh from Rana chensinensis, which has 12-15 amino acid residues in its mature peptide region, Temporin-1Ja consists of 13 amino acids . Additionally, Temporin-1Ja has distinct hydrophobicity and net charge characteristics compared to other temporins, lacking the cysteine residues found in some other antimicrobial peptides like japonicin family members (japonicin-1Ja: FFPIGVFCKIFKTC and japonicin-2Ja: FGLPMLSILPKALCILLKRKC) from the same species .

What is the biosynthetic precursor structure for Temporin-1Ja?

The biosynthetic precursor of Temporin-1Ja follows the typical organization of amphibian antimicrobial peptide precursors. Based on molecular cloning studies, the precursor contains:

• A highly conserved signal peptide region

• A spacer or intervening region with relatively well-conserved sequences

• A characteristic -Lys-Arg- propeptide convertase cleavage site

• The hypervariable mature peptide region (Temporin-1Ja)

• A C-terminal glycine residue that serves as an amide donor for C-terminal amidation

This structural organization is conserved across temporin family peptides from different Rana species. The precursor undergoes post-translational processing including proteolytic cleavage at the -Lys-Arg- site and C-terminal amidation to yield the mature, biologically active Temporin-1Ja peptide . The molecular cloning of cDNAs encoding Temporin-1Ja from R. japonica skin has confirmed this precursor structure, with significant similarities to other temporin precursors from related species .

What molecular cloning techniques are most effective for isolating Temporin-1Ja cDNA from Rana japonica?

The most effective molecular cloning approach for isolating Temporin-1Ja cDNA from Rana japonica involves a combination of reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). This methodology has been successfully demonstrated in research studies:

• RNA Extraction: Total RNA is isolated from Rana japonica skin, the primary tissue expressing Temporin-1Ja. Using TRIzol reagent or similar RNA extraction methods yields high-quality RNA suitable for downstream applications.

• RT-PCR with Degenerate Primers: Initial amplification employs degenerate primers designed based on conserved regions of known temporin family precursors, particularly targeting the signal peptide region which shows high conservation across amphibian antimicrobial peptides.

• 5'- and 3'-RACE: To obtain complete cDNA sequences, both 5' and 3' RACE techniques are employed to amplify the full-length cDNA including untranslated regions. This step is crucial for characterizing the complete precursor structure.

• Cloning and Sequencing: Amplified products are cloned into appropriate vectors (such as pGEM-T Easy) and sequenced to confirm identity and accuracy.

This combinatorial approach has successfully yielded cDNAs encoding the biosynthetic precursor for Temporin-1Ja along with other antimicrobial peptides like japonicin-1Ja and japonicin-2Ja from R. japonica skin total RNA preparations . The effectiveness of this approach is evidenced by successful characterization of the complete precursor structure, including signal peptide, intervening sequence, and mature peptide regions.

Which expression systems are optimal for producing recombinant Temporin-1Ja?

Several expression systems can be employed for recombinant production of Temporin-1Ja, each with distinct advantages depending on research objectives:

• Bacterial Expression Systems (E. coli):

  • Most commonly used due to simplicity, cost-effectiveness, and high yield

  • Expression strategies typically involve:
    a) Fusion protein approaches using tags like thioredoxin, SUMO, or GST to improve solubility and reduce toxicity to host cells
    b) Periplasmic expression to facilitate proper folding

    • Cleavage sites (enzymatic or chemical) must be engineered for tag removal

  • Limitations include potential endotoxin contamination and lack of post-translational modifications

• Yeast Expression Systems (P. pastoris, S. cerevisiae):

  • Advantages include eukaryotic protein processing capabilities and secretion

  • More suitable for temporins requiring post-translational modifications like C-terminal amidation

  • Lower endotoxin concerns compared to bacterial systems

• Cell-Free Expression Systems:

  • Allow direct synthesis of potentially toxic peptides without cellular viability concerns

  • Particularly valuable for temporins with antimicrobial activity that might inhibit host cell growth

  • Enable rapid production for screening studies

For Temporin-1Ja specifically, bacterial expression systems using fusion partners have shown success, though special consideration must be given to the C-terminal amidation requirement, which often necessitates chemical synthesis steps following recombinant production. The choice between systems depends on research needs regarding yield, purity, post-translational modifications, and downstream applications .

What are the key challenges in achieving proper folding and post-translational modifications in recombinant Temporin-1Ja?

Producing correctly folded and post-translationally modified recombinant Temporin-1Ja presents several key challenges:

• C-terminal Amidation:

  • Temporin-1Ja naturally contains an amidated C-terminus (ILPLVGNLLNDLL.NH₂)

  • Most recombinant expression systems lack the enzymatic machinery for this modification

  • Solutions include:
    a) Enzymatic amidation following purification using peptidyl-glycine α-amidating monooxygenase
    b) Chemical amidation strategies
    c) Synthetic approaches like solid-phase peptide synthesis (SPPS) as demonstrated with temporin-1CEh

• Preserving α-helical Structure:

  • Temporin-1Ja adopts α-helical conformation in membrane environments critical for activity

  • Expression conditions affecting proper folding include:
    a) pH of culture medium and purification buffers
    b) Ionic strength of solutions
    c) Presence of structure-stabilizing agents

• Avoiding Proteolytic Degradation:

  • Short antimicrobial peptides are susceptible to proteolytic degradation

  • Strategies include:
    a) Use of protease-deficient host strains
    b) Inclusion of protease inhibitors during purification
    c) Expression as fusion proteins to shield from proteolysis

• Cytotoxicity to Host Cells:

  • Antimicrobial activity can affect host cell viability

  • Approaches include:
    a) Inducible expression systems with tight regulation
    b) Secretion strategies to remove peptide from cytoplasm
    c) Fusion with neutralizing domains

Researchers have addressed these challenges through combined recombinant and synthetic approaches, where recombinant systems produce the unmodified peptide backbone, followed by chemical methods to achieve proper post-translational modifications .

What is the antimicrobial spectrum of Temporin-1Ja compared to other temporin family peptides?

Temporin-1Ja demonstrates a distinctive antimicrobial spectrum compared to other temporin family members, showing activity primarily against Gram-positive bacteria with limited effectiveness against Gram-negative bacteria. This antimicrobial profile can be compared with other temporins as follows:

The temporin family generally shows stronger activity against Gram-positive bacteria, with the notable exception of P. melaninogenica among Gram-negative species. Temporin-1DRa and Temporin-1Va are particularly promising as drug development candidates due to their relatively low hemolytic activity against human erythrocytes compared to melittin-related peptides . Modified temporins, such as engineered variants of Temporin-1CEh (T1CEh-KKPWW), have been designed to achieve broader antimicrobial spectrum including enhanced activity against Gram-negative bacteria .

How does the disulfide bond status affect antimicrobial activity in temporin family peptides?

The presence or absence of disulfide bonds significantly impacts the antimicrobial activity of temporin family peptides, though Temporin-1Ja itself does not contain cysteine residues for disulfide bond formation. Research on related peptides from Rana japonica provides valuable insights:

• Temporin-1Ja (ILPLVGNLLNDLL.NH₂):

  • Contains no cysteine residues

  • Maintains linear conformation without disulfide bonds

  • Activity depends on adopting α-helical structure in membrane environments

  • Lacks the structural stabilization provided by disulfide bonds

• Japonicin Family Comparison:

  • Japonicin-2Ja (FGLPMLSILPKALCILLKRKC) from R. japonica contains cysteine residues

  • Antimicrobial assays comparing cyclic (disulfide-bonded) and linear japonicin-2Ja demonstrate that "the intramolecular disulfide bond is necessary for activity"

  • This provides comparative context for understanding structural requirements within R. japonica antimicrobial peptides

• Functional Implications:

  • Disulfide bonds provide:
    a) Increased structural stability
    b) Resistance to proteolytic degradation
    c) Constrained conformations that may enhance target interactions
    d) Potential for redox-dependent activity modulation

• Structure-Activity Relationships:

  • Linear temporins rely on environment-induced folding for activity

  • Disulfide-containing peptides maintain more rigid conformations

  • Different structural constraints affect:
    a) Membrane penetration ability
    b) Target selectivity
    c) Stability in physiological conditions

These structure-activity relationships are important considerations when designing modified temporins for enhanced antimicrobial properties. The comparative analysis between linear Temporin-1Ja and disulfide-containing antimicrobial peptides from the same species provides valuable insights into structural determinants of activity .

What are the expression patterns of Temporin-1Ja across different tissues in Rana japonica?

The expression of Temporin-1Ja varies significantly across different tissues in Rana japonica, with the highest expression observed in skin tissue. Semi-quantitative analysis using real-time RT-PCR with TaqMan probes has revealed the following relative expression patterns for the preprotemporin-1Ja mRNA:

This expression profile demonstrates that skin tissue exhibits dramatically higher expression levels (approximately 65,000 times higher than stomach tissue), consistent with the primary defensive role of antimicrobial peptides in amphibian skin. The expression in other tissues, while present, is orders of magnitude lower than in skin . This tissue-specific expression pattern aligns with the primary function of temporins as components of the innate immune defense system in the integument, which serves as the first line of defense against environmental pathogens.

What structure-activity relationships have been identified for Temporin-1Ja and related peptides?

Structure-activity relationships (SARs) for Temporin-1Ja and related temporin peptides reveal critical molecular features that determine their antimicrobial activity, selectivity, and potential therapeutic applications:

• Peptide Length:

  • Temporin-1Ja's 13-amino acid length falls within the optimal range (10-14 residues) for temporin activity

  • Shorter peptides generally struggle to span bacterial membranes

  • Longer peptides may have reduced membrane penetration ability

• Amphipathicity and Helicity:

  • The ability to form amphipathic α-helices correlates strongly with antimicrobial activity

  • Hydrophobic moment (a measure of amphipathicity) is a key predictor of membrane interaction

  • Temporal separation of hydrophobic and hydrophilic faces enables membrane disruption

• Net Charge:

  • Modest positive charge facilitates initial electrostatic interaction with negatively charged bacterial membranes

  • Charge modifications in temporin analogs significantly impact activity spectrum:
    a) Increased positive charge enhances activity against Gram-negative bacteria
    b) The T1CEh-KKPWW analog (with added lysine residues) showed potent activity against Gram-negative bacteria in vitro and in vivo

• Hydrophobicity:

  • Optimal hydrophobicity balance is crucial:
    a) Too hydrophobic: increases hemolytic activity and reduces selectivity
    b) Too hydrophilic: reduces membrane penetration and antimicrobial activity

  • Leucine content in Temporin-1Ja contributes significantly to its hydrophobicity

• Terminal Modifications:

  • C-terminal amidation (present in Temporin-1Ja) typically enhances:
    a) Stability against carboxypeptidases
    b) Increased positive charge
    c) Improved membrane interaction

• Strategic Substitutions:

  • Studies with temporin-1CEh demonstrated that strategic substitutions can dramatically alter activity:
    a) Addition of tryptophan residues enhances membrane interaction
    b) Lysine substitutions increase positive charge and antimicrobial spectrum
    c) Proline insertions affect helicity and membrane penetration

These structure-activity relationships provide the foundation for rational design of temporin analogs with enhanced antimicrobial properties and reduced toxicity to mammalian cells, as demonstrated with the development of T1CEh-KKPWW and other engineered temporin variants .

What approaches have been successful for designing improved temporin analogs with enhanced antimicrobial properties?

Several successful approaches have been employed to design improved temporin analogs with enhanced antimicrobial properties, based on rational modification strategies informed by structure-activity relationships:

• Charge Modification Strategies:

  • Addition of positively charged residues (typically lysine or arginine)

  • Example: T1CEh-KKPWW incorporated additional lysine residues, resulting in:
    a) "Potent antibacterial activity with significantly increasing the activity against Gram-negative bacteria in vitro and in vivo"
    b) Maintained low hemolytic activity

• Hydrophobicity Adjustment:

  • Strategic replacement of amino acids to optimize hydrophobic/hydrophilic balance

  • Addition of tryptophan residues enhances membrane interaction due to their ability to position at the membrane-water interface

• Secondary Structure Modification:

  • Introduction of helix-promoting or helix-breaking residues

  • Proline insertions can create kinks that affect membrane interaction patterns

  • Maintenance of amphipathic helical structure through careful substitution patterns

• Branched Peptide Design:

  • Novel branched structures allow active units to accumulate

  • Example: T1CEh-KKPWW2 demonstrated:
    a) High sensitivity to pH, serum, or salt conditions
    b) Stable antibacterial activity despite changing conditions
    c) Potential as "a candidate in future antimicrobial and antibiofilm applications"

• Terminal Modifications:

  • Beyond the natural C-terminal amidation:
    a) N-terminal acetylation to increase stability
    b) Addition of hydrophobic caps to enhance membrane penetration
    c) Lipidation to anchor peptides in membranes

• Hybrid Peptide Approaches:

  • Combination of temporin sequence elements with components from other antimicrobial peptide families

  • Creation of chimeric peptides with broader spectrum activity

• Cyclization Strategies:

  • Introduction of constraints through:
    a) Disulfide bridges
    b) Lactam bridges
    c) Stapling technologies

These design strategies have yielded temporin analogs with enhanced properties, including broader antimicrobial spectrum, improved stability under physiological conditions, reduced susceptibility to proteolytic degradation, and maintained selectivity (antimicrobial vs. hemolytic activity). The successful engineering of T1CEh-KKPWW and T1CEh-KKPWW2 variants demonstrates the potential of these approaches for developing temporin-based antimicrobials with improved therapeutic potential .

How can recombinant expression systems be optimized for the production of Temporin-1Ja analogs?

Optimizing recombinant expression systems for Temporin-1Ja analogs requires addressing several key challenges to achieve high yield, proper folding, and necessary post-translational modifications. The following strategies represent the most effective approaches:

• Fusion Partner Optimization:

  • Selection of appropriate fusion tags based on specific properties:
    a) Thioredoxin (Trx): Enhances solubility and reduces toxicity
    b) SUMO: Facilitates proper folding and provides clean cleavage
    c) Ubiquitin: Improves expression and provides native N-terminus after cleavage
    d) KSI (ketosteroid isomerase): Directs peptide to inclusion bodies, protecting host cells

  • Incorporation of precision cleavage sites (TEV protease, SUMO protease, or Factor Xa) for tag removal

• Codon Optimization:

  • Adaptation of codons to expression host preference:
    a) Avoidance of rare codons
    b) Optimization of GC content
    c) Elimination of unwanted secondary structures in mRNA

  • Implementation of tandem repeats for multi-peptide expression from single construct

• Expression Host Selection:

  • For Temporin-1Ja analogs requiring C-terminal amidation:
    a) Mammalian cells with α-amidating enzyme co-expression
    b) Insect cells with natural amidation capability
    c) Engineered yeast strains with introduced amidation pathways

  • For analogs with disulfide bonds:
    a) Bacterial strains with enhanced disulfide bond formation (Origami, SHuffle)
    b) Periplasmic expression strategies

• Induction Strategy Refinement:

  • Temperature optimization: Lower temperatures (15-25°C) often improve folding

  • Inducer concentration tuning: Lower concentrations can reduce inclusion body formation

  • Induction timing: Induction at higher cell densities can improve yield

  • Extended expression times at reduced temperatures

• Medium and Culture Condition Optimization:

  • Auto-induction media to eliminate manual induction steps

  • Supplementation with amino acids for challenging peptides

  • Adjustment of aeration and pH conditions

• Harvest and Purification Strategy:

  • For inclusion body-based expression:
    a) Specialized solubilization buffers
    b) Refolding protocols optimized for antimicrobial peptides

  • For soluble expression:
    a) Gentle lysis conditions
    b) Protease inhibitor cocktails
    c) Immediate pH adjustment to prevent degradation

• Scale-Up Considerations:

  • Bioreactor parameters optimization:
    a) Dissolved oxygen control
    b) Feed strategy development
    c) pH control systems

These optimization strategies must be tailored to the specific properties of each Temporin-1Ja analog, considering factors such as charge, hydrophobicity, and structural requirements. For complex modifications requiring precise post-translational processing, combined approaches utilizing recombinant expression followed by chemical modification may be most effective, as demonstrated with temporin-1CEh analogs synthesized using solid-phase peptide synthesis methods .

How can recombinant Temporin-1Ja be utilized in studying bacterial membrane interactions?

Recombinant Temporin-1Ja provides a powerful tool for investigating bacterial membrane interactions through various experimental approaches:

• Fluorescently Labeled Peptide Studies:

  • Site-specific labeling of recombinant Temporin-1Ja enables:
    a) Real-time visualization of membrane binding and penetration
    b) Quantification of membrane localization
    c) Fluorescence resonance energy transfer (FRET) studies to measure peptide-peptide interactions during membrane disruption

• Membrane Model Systems:

  • Recombinant Temporin-1Ja can be studied with:
    a) Liposomes of varying lipid compositions to mimic different bacterial membranes
    b) Giant unilamellar vesicles (GUVs) for single-vesicle visualization
    c) Supported lipid bilayers for atomic force microscopy analyses

  • These systems allow controlled investigation of:
    a) Lipid preference and specificity
    b) Critical peptide concentration thresholds
    c) Membrane disruption kinetics

• Biophysical Characterization Techniques:

  • Circular dichroism (CD) spectroscopy to monitor secondary structure changes upon membrane binding

  • Nuclear magnetic resonance (NMR) studies to determine peptide orientation in membranes

  • Differential scanning calorimetry to measure thermodynamic effects on membrane properties

• Genetic Reporter Systems:

  • Construction of bacterial strains with reporters for:
    a) Membrane potential disruption
    b) Membrane permeability changes
    c) Stress response activation

  • These systems enable high-throughput screening of Temporin-1Ja variants

• Real-Time Permeabilization Assays:

  • Dye leakage assays from liposomes

  • Propidium iodide uptake in bacterial cells

  • Membrane depolarization using potential-sensitive dyes

  • These approaches provide quantitative measures of membrane disruption kinetics

• Comparative Studies with Structure-Function Analysis:

  • Using recombinant Temporin-1Ja alongside engineered variants to establish:
    a) Key residues for membrane binding
    b) Critical structural elements for pore formation
    c) Determinants of selectivity between bacterial and mammalian membranes

These research applications offer mechanistic insights into how Temporin-1Ja and related antimicrobial peptides interact with bacterial membranes, potentially leading to the design of more effective antimicrobial agents. The membrane-targeting mechanism of temporins, which has been demonstrated to act through membrane destruction for peptides like Temporin-1CEh, provides an important model system for studying antimicrobial peptide action more broadly .

What are the most promising research directions for Temporin-1Ja in combating antibiotic-resistant bacteria?

Research on Temporin-1Ja presents several promising directions for addressing antibiotic resistance, leveraging its membrane-targeting mechanism and potential for synthetic optimization:

• Synergistic Combination Therapies:

  • Investigation of Temporin-1Ja in combination with:
    a) Conventional antibiotics to enhance penetration through bacterial membranes
    b) Other antimicrobial peptides with complementary mechanisms
    c) Membrane-permeabilizing agents to achieve synergistic effects

  • These approaches may allow lower effective doses and reduced resistance development

• Anti-Biofilm Applications:

  • Exploration of Temporin-1Ja's potential against bacterial biofilms:
    a) Biofilm penetration capabilities
    b) Activity against metabolically inactive persister cells
    c) Disruption of biofilm matrix components

  • Studies with temporin family peptides have shown promising anti-biofilm activity, suggesting similar potential for Temporin-1Ja variants

• Engineered Delivery Systems:

  • Development of:
    a) Nanoparticle encapsulation to enhance stability and targeting
    b) Stimuli-responsive release systems activated at infection sites
    c) Surface-immobilized peptides for antimicrobial surfaces and devices

  • These systems could overcome delivery challenges while maintaining antimicrobial efficacy

• Rational Design of Low-Resistance Variants:

  • Engineering Temporin-1Ja analogs with:
    a) Multiple simultaneous membrane-disruption mechanisms
    b) Rapid killing kinetics to limit resistance development
    c) Reduced susceptibility to proteolytic degradation

  • The T1CEh-KKPWW analog demonstrates how engineering can enhance activity against both Gram-positive and Gram-negative bacteria

• Structure-Guided Optimization for ESKAPE Pathogens:

  • Targeted development against priority antibiotic-resistant pathogens:
    a) Pseudomonas aeruginosa
    b) Acinetobacter baumannii
    c) Enterobacteriaceae with carbapenem resistance

  • Some temporin family peptides have shown activity against clinical isolates of methicillin-resistant Staphylococcus aureus, suggesting potential against resistant strains

• Immunomodulatory Applications:

  • Investigation of Temporin-1Ja's potential to:
    a) Modulate host immune responses to infection
    b) Enhance neutrophil chemotaxis
    c) Influence inflammatory cytokine production

  • These immunomodulatory effects could complement direct antimicrobial activity

The membrane-targeting mechanism of Temporin-1Ja makes it particularly promising against antibiotic-resistant bacteria, as this mode of action is difficult for bacteria to develop resistance against compared to conventional antibiotics with specific molecular targets. The successful engineering of temporin analogs with enhanced properties, such as the broad-spectrum activity demonstrated by T1CEh-KKPWW, provides a roadmap for similar optimization of Temporin-1Ja .

What methodological advances are needed to fully characterize the in vivo efficacy and safety of recombinant temporins?

Fully characterizing the in vivo efficacy and safety of recombinant temporins, including Temporin-1Ja, requires several methodological advances across different research domains:

• Advanced Animal Infection Models:

  • Development of:
    a) Immunocompromised models that better mimic human susceptibility
    b) Tissue-specific infection models (skin, respiratory, urinary tract)
    c) Chronic infection models for persistent pathogens
    d) Biofilm-associated infection models

  • These would provide more clinically relevant efficacy assessments than current simplistic models

• Pharmacokinetic and Pharmacodynamic (PK/PD) Methodologies:

  • Improved techniques for:
    a) Tracking peptide distribution in tissues with high sensitivity
    b) Determining bioavailability after various administration routes
    c) Assessing plasma protein binding effects
    d) Measuring peptide stability in biological fluids

  • Current limitations in peptide detection sensitivity and specificity hinder comprehensive PK/PD analysis

• Immunogenicity Assessment Platforms:

  • More sophisticated methods to evaluate:
    a) Antibody development against recombinant temporins
    b) T-cell responses to repeated administration
    c) Potential for hypersensitivity reactions
    d) Long-term immunological memory effects

• Resistance Development Monitoring:

  • Standardized protocols for:
    a) Long-term resistance evolution studies
    b) Cross-resistance assessment between different AMPs
    c) Genomic and transcriptomic analysis of exposed bacterial populations
    d) Membrane composition adaptation monitoring

• Toxicity Evaluation Methods:

  • Beyond hemolysis assays, development of:
    a) Membrane selectivity indices based on multiple cell types
    b) Organ-specific toxicity assessments
    c) Local tissue irritation quantification methods
    d) Long-term exposure safety studies

• Delivery System Integration:

  • Methods to assess:
    a) In vivo release kinetics from delivery systems
    b) Targeted delivery efficiency to infection sites
    c) Stability enhancement through formulation
    d) Tissue penetration in complex infection environments

• Standardized Production and Quality Control:

  • Development of: a) Validated analytical methods for recombinant temporin characterization b) Reference standards for activity comparison c) Impurity profiling specific to recombinant peptides d) Batch-to-batch consistency assessment protocols