Recombinant Kalata-B14

What is Kalata-B14 and what is its natural source?

Kalata-B14 is a cyclotide originally isolated from the plant Oldenlandia affinis, which belongs to the Rubiaceae family. This plant has been traditionally used in various cultures for its medicinal properties, which are partially attributed to the presence of cyclotides like Kalata-B14. Kalata-B14 belongs to the "kalata" subfamily of cyclotides, characterized by a specific arrangement of amino acids and disulfide bonds that provide exceptional stability and bioactivity.

How does the structure of Kalata-B14 compare with other cyclotides from O. affinis?

Kalata-B14 shares the distinctive cyclic cystine knot (CCK) motif common to all cyclotides. Similar to Kalata B1 and B2 (which account for approximately 34% of the cyclotide content in O. affinis), Kalata-B14 has a cyclic backbone structure stabilized by three disulfide bonds . While Kalata B1 and B2 differ by only five amino acid positions with minimal structural consequences (backbone RMSD of 0.599 Å), Kalata-B14 has its own unique amino acid sequence while maintaining the characteristic cyclotide scaffold . The molecular formula of Kalata-B14 is C₆₄H₁₀₆N₁₈O₁₇S₃, with a molecular weight of approximately 1,258 Da.

What biological activities have been documented for Kalata cyclotides?

Cyclotides from O. affinis, including Kalata variants, demonstrate diverse biological activities. Research has documented:

• Immunosuppressive properties: Kalata B1 shows dose-dependent antiproliferative effects on primary activated human lymphocytes with an IC₅₀ of 3.9 ± 0.5 μM

• Insecticidal and nematocidal activities

• Antifouling properties

• Anti-HIV activity

• Cytotoxicity against lymphoma cell lines

• Uterotonic activity (ability to induce uterine contractions)

These activities are generally attributed to the unique structural features of cyclotides, particularly their cyclic backbone and disulfide bond arrangement.

What expression systems are most suitable for recombinant Kalata-B14 production?

Based on successful approaches with related cyclotides, bacterial expression systems using Escherichia coli represent the most well-established platform for recombinant cyclotide production. The specific methodology involves:

• Cloning a synthetic codon-optimized cDNA sequence encoding the cyclotide

• Creating a fusion construct with a carrier protein (e.g., thioredoxin with a His₆-tag)

• Expression in a suitable E. coli strain such as BL21(DE3)

• Purification using affinity chromatography (e.g., metal-chelating Sepharose column)

• Release of the target peptide using specific proteases

For Kalata B1, this approach has demonstrated efficient production of properly folded peptides with correct disulfide bond formation, suggesting its applicability to Kalata-B14 .

How can intein-based systems be employed for cyclotide production?

Recent advances in cyclotide production have utilized conditional intein systems, which offer significant advantages for recombinant cyclotide synthesis:

• System design principles:

  • Implementation of promiscuous extein recognition sites allowing cyclization of virtually any peptide

  • Incorporation of a secondary split site within the intein enabling triggered splicing

  • Expression of two separate intein precursors that can self-assemble in vitro

• Production workflow:

  • Recombinant expression and purification of intein precursors

  • In vitro self-assembly to facilitate cyclization

  • Purification of the cyclized product

  • Proper folding to establish correct disulfide bond formation

This approach has been validated with Kalata B1, resulting in a product with identical structure to synthetic versions and retaining full biological activity as demonstrated in insect cell toxicity assays .

What strategies ensure proper folding and disulfide bond formation?

Correct disulfide bond formation is critical for cyclotide bioactivity. Methodological approaches include:

• Oxidative folding conditions: Optimized buffer systems containing redox pairs (e.g., reduced/oxidized glutathione) that facilitate proper disulfide formation

• Temperature control: Thermal stability studies have demonstrated that recombinant acyclic Kalata B1 exhibits exceptional stability against thermal denaturation

• Oxidation conditions: Hydrogen peroxide has been found to enhance proteolytic cleavage rates in a concentration-dependent manner during processing

• Structural validation: Mass spectrometry and NMR analysis to confirm correct disulfide bond formation and proper three-dimensional structure

What techniques are essential for confirming cyclotide structure and purity?

Multiple complementary analytical techniques are required for comprehensive characterization:

• Mass spectrometry:

  • Electrospray ionization mass spectrometry (ESI-MS) for molecular weight confirmation

  • Tandem MS (MS/MS) for sequence verification

  • Mass spectrometric analysis to confirm oxidation state (fully oxidized form indicates proper disulfide bond formation)

• NMR spectroscopy:

  • 2D NMR for structural confirmation

  • Comparison with reference spectra of synthetic or native cyclotides to verify correct folding

• Chromatographic methods:

  • Reversed-phase HPLC for final purification and purity assessment

  • Size-exclusion chromatography to separate cyclized product from linear precursors

How can researchers differentiate between correctly folded and misfolded cyclotides?

Distinguishing properly folded cyclotides requires multi-parameter analysis:

• Bioactivity assays: Comparison of biological activity with synthetic or native standards (e.g., insect cell toxicity assays for Kalata B1)

• Thermal stability analysis: Properly folded cyclotides show exceptional resistance to thermal denaturation

• Disulfide bond mapping: Chemical or enzymatic approaches to confirm correct disulfide connectivity

• Circular dichroism (CD) spectroscopy: To assess secondary structure elements characteristic of properly folded cyclotides

What cellular assays are appropriate for evaluating Kalata-B14 immunosuppressive activity?

Based on studies with Kalata B1, the following methodological approaches are recommended:

• Cell proliferation assays:

  • CFSE (carboxyfluorescein succinimidyl ester) assay to measure cell division capacity of primary activated peripheral blood mononuclear cells (PBMCs)

  • Determine dose-response relationships and calculate IC₅₀ values

  • Evaluate concentration ranges between 1.8-14 μM based on Kalata B1 data

• Cytotoxicity differentiation:

  • Apoptosis detection: Internucleosomal DNA fragmentation (subG1 analysis)

  • Phosphatidylserine surface analysis through annexin V and propidium iodide staining

  • Forward-side-scatter cytometric analysis to distinguish cytostatic effects from cytotoxicity

• Control experiments:

  • Include positive controls for apoptosis (e.g., CPT at 30 μg/mL)

  • Include positive controls for necrosis (e.g., Triton-X 100)

  • Include membrane-disrupting peptides (e.g., melittin) as comparators

How should researchers interpret dose-dependent effects of cyclotides?

Interpretation requires careful analysis across multiple concentrations:

• Concentration-specific effects: For Kalata B1, concentrations between 1.8-14 μM produce dose-dependent antiproliferative effects without significant cytotoxicity, while concentrations above 14 μM become cytotoxic to cells

• Mechanism differentiation:

  • Cytostatic vs. cytotoxic effects require different experimental endpoints

  • Hemolysis and membrane disruption typically occur at higher concentrations (>50 μM for Kalata B1)

• Data integration:

  • Correlate proliferation data with cytotoxicity measurements

  • Consider timing of effects (immediate vs. delayed responses)

  • Compare with known reference compounds having established mechanisms of action

What are common challenges in recombinant cyclotide expression and how can they be addressed?

Several technical challenges may arise during recombinant cyclotide production:

• Poor expression yields:

  • Solution: Optimize codon usage for the host organism

  • Solution: Test different fusion partners (thioredoxin has shown success)

  • Solution: Evaluate different E. coli strains optimized for disulfide bond formation

• Inefficient proteolytic release:

  • Solution: Incorporate an enterokinase recognition sequence immediately upstream of the target peptide

  • Solution: Optimize hydrogen peroxide concentration to enhance cleavage rates

  • Solution: Explore alternative proteases with higher specificity

• Incorrect folding:

  • Solution: Implement optimized oxidative folding conditions

  • Solution: Consider co-expression with disulfide isomerases

  • Solution: Explore refolding from inclusion bodies under controlled conditions

How can researchers optimize purification of recombinant cyclotides?

Efficient purification strategies include:

• Multi-step purification:

  • Initial capture using affinity chromatography (e.g., metal-chelating Sepharose for His-tagged constructs)

  • Separation of cleaved target peptide from carrier protein using ultracentrifugation through semipermeable membranes

  • Final purification via reversed-phase HPLC

• Scaling considerations:

  • Implement tangential flow filtration for larger-scale preparations

  • Consider ion exchange chromatography as an orthogonal purification step

  • Optimize buffer conditions to maximize yield and purity

How can recombinant Kalata-B14 be engineered for enhanced immunosuppressive properties?

Structure-function relationship studies suggest several strategies:

• Site-directed mutagenesis:

  • Target amino acid positions that differ between Kalata B1 and B2, which show similar bioactivity profiles despite sequence differences

  • Modify surface-exposed residues involved in membrane interactions

  • Introduce non-natural amino acids at key positions to enhance stability or target binding

• Loop engineering:

  • Cyclotides contain six loops between cysteine residues that can be modified while maintaining the core scaffold

  • Systematic substitutions in specific loops can enhance target selectivity and potency

  • Grafting of bioactive sequences from other peptides into cyclotide loops

What emerging technologies might improve recombinant Kalata-B14 production?

Recent advances offer new possibilities for cyclotide engineering:

• Conditional intein systems:

  • Implementation of promiscuous extein recognition sites

  • Secondary split sites enabling triggered splicing

  • In vitro self-assembly of intein precursors

• Cell-free expression systems:

  • Rapid prototyping of cyclotide variants

  • Elimination of cell viability concerns for toxic peptides

  • Direct incorporation of non-canonical amino acids

• Non-bacterial expression hosts:

  • Exploration of yeast or plant-based expression systems

  • Potential for glycoengineering or other post-translational modifications

  • Scale-up potential in bioreactor systems