Recombinant Kalata-B10

What is the basic structure of Kalata B1 and what makes it uniquely stable?

Kalata B1 is a cyclic peptide characterized by a head-to-tail cyclized backbone and a cystine knot core. These structural features contribute to its exceptional stability under extreme conditions. Research has demonstrated that Kalata B1 remains stable when exposed to chaotropic agents such as 6 M guanidine hydrochloride and 8 M urea, temperatures approaching boiling, acidic conditions, and various proteases . The cystine knot structure has been identified as more critical than the circular backbone for maintaining chemical stability, as evidenced by comparative studies with acyclic permutants and a two-disulfide derivative .

How can the structural characteristics of Kalata B1 be analyzed using NMR spectroscopy?

NMR spectroscopy provides detailed insights into Kalata B1's structural characteristics. Specifically, researchers use 1H-1H coupling constants (3JNH-CαH) and techniques such as DQFC and TOCSY to complete chemical shift assignments of side chains . For the three proline residues at positions 3, 17, and 24, their isomeric states (cis or trans) can be identified through NOE cross peaks. Pro 3 and Pro 17 exist as trans isomers, indicated by NOE cross peaks between the Hδ of the proline and the Hα of the preceding residue. Conversely, Pro 24 exists as a cis isomer, evidenced by the NOE cross peak between the Hα of the proline and another residue .

What are the typical methods for confirming proper folding and structure of recombinantly produced Kalata B1?

Recombinantly produced Kalata B1 requires rigorous characterization to confirm proper folding and structure. This typically involves:

• Mass spectrometry to confirm cyclization and correct molecular weight

• NMR spectroscopy for structural analysis and comparison with synthetic standards

• Disulfide bond formation verification

• Biological activity assays (such as insect cell toxicity) to confirm functional equivalence

Research has demonstrated that properly cyclized and folded recombinant Kalata B1 exhibits identical structure to synthetic Kalata B1 as determined by mass spectrometry and NMR, with correct disulfide bond formation .

What is the conditional intein system for recombinant production of Kalata B1?

The conditional intein system represents a significant advancement in recombinant cyclotide production. This system is based on two key features:

• A promiscuous extein recognition site that allows cyclization of virtually any peptide sequence

• A secondary split site within the intein itself that enables triggered splicing at will

In practical implementation, two intein precursors are recombinantly expressed separately, purified, and then self-assembled in vitro to cyclize Kalata B1. This controlled approach ensures efficient cyclization under specific conditions, providing researchers with precise control over the production process .

What expression systems work best for recombinant Kalata B1 production?

While specific expression systems were not extensively detailed in the provided research, successful recombinant production of Kalata B1 has been achieved through:

• Separate expression of intein precursors

• Purification of these components

• In vitro self-assembly under controlled conditions

This approach circumvents potential toxicity issues that might arise from expressing the active cyclotide directly in host cells. The methodology appears particularly suited for bacterial expression systems, though optimization would be required for each specific cyclotide variant.

What purification strategies are most effective for recombinant Kalata B1?

Effective purification of recombinant Kalata B1 involves multiple steps:

• Initial purification of intein precursors (likely using affinity chromatography)

• In vitro cyclization reaction

• Purification of the cyclized product

• Refolding to ensure proper disulfide bond formation

The specific techniques may include HPLC for final purification, with subsequent characterization by mass spectrometry and NMR to confirm proper structure and folding .

How does linearization affect Kalata B1's biological activities?

Most significantly, linearization maintains the basic structure but eliminates hemolytic activity, indicating that the circular backbone is critical for certain biological functions despite not being the primary determinant of chemical stability .

What structural elements determine Kalata B1's membrane interactions?

Additionally, experiments with synthetic all-D-isomers of Kalata B1 showed equivalent activity to the naturally occurring L-isomer, further confirming that there is no chiral requirement for activity. This lack of chiral selectivity supports the proposed mode of action involving membrane-based interactions rather than specific receptor binding .

How do the structures of natural and synthetic Kalata B1 compare in terms of biological activity?

Comparative studies of natural and synthetic Kalata B1 reveal remarkable similarity in both structure and function. Natural Kalata B1 demonstrated an IC50 value of 7.9 μM in U-87 MG cells, comparable to synthetic Kalata B1's IC50 value of 5.4 μM in the same cell line . The biological activity profiles are similarly aligned across different cell types, suggesting that synthetic versions accurately replicate the structural features essential for activity .

How can Kalata B1 be utilized as a chemosensitizer for glioblastoma treatment?

Recent research has demonstrated Kalata B1's potential as a chemosensitizer for temozolomide (TMZ) in glioblastoma treatment. When glioblastoma cells (U-87 MG) were co-exposed to synthetic Kalata B1 (0.5 μM) and TMZ, a 16-fold lower concentration of TMZ (100 μM) was sufficient to achieve significant cytotoxicity . Similarly, in T-98 cells, co-exposure with synthetic Kalata B1 (0.25 μM) reduced the required TMZ concentration by 15-fold (to 75 μM) .

This chemosensitization effect represents a promising avenue for overcoming resistance to standard treatments in glioblastoma therapy. The exact mechanism may involve Kalata B1's membrane-disrupting capabilities, which could enhance TMZ uptake or retention in cancer cells .

What methods can be used to assess Kalata B1 stability in physiological conditions?

Evaluation of Kalata B1 stability under physiological conditions is critical for therapeutic applications. Human serum stability assays provide valuable insights:

• Stock synthetic Kalata B1 (300 μM) is diluted 10 times with warmed plasma or PBS

• Samples are incubated for various timepoints (0, 3, 8, and 24 h)

• Controls consist of Kalata B1 in PBS incubated in parallel

• Serum proteins are denatured with urea (3M final concentration) at 4°C for 10 min

• Trichloroacetic acid (7% final v/v) is added to precipitate proteins

• Samples are centrifuged at 15,000× g for 10 min

• The supernatant is analyzed using UPLC-MS

This methodology has confirmed Kalata B1's remarkable stability in human serum, a critical attribute for potential therapeutic applications .

What is the relative importance of the cystine knot versus circular backbone for Kalata B1 stability?

Comparative studies of Kalata B1 variants have provided crucial insights into the structural determinants of stability. Research utilizing several Kalata B1 variants—including Kalata B2 (with five amino acid substitutions), acyclic permutants (with broken backbone but intact cystine knot), and a two-disulfide bond mutant—has elucidated the relative contributions of different structural elements .

The addition of denaturants to native Kalata B1 or acyclic permutants did not cause unfolding, while the two-disulfide derivative showed reduced stability despite having a similar three-dimensional structure. This evidence strongly suggests that the cystine knot is more critical than the circular backbone for the chemical stability of cyclotides . Furthermore, the cystine knot in cyclotides appears more stable than those in similar-sized molecules, as demonstrated by comparisons with conotoxin PVIIA .

What IC50 determination methods are most reliable for Kalata B1 cytotoxicity studies?

For reliable IC50 determination in cytotoxicity studies, the following methodology has been validated:

• Cell viability assays using appropriate cell lines (e.g., U-87 MG, T-98G)

• Dose-dependent inhibition analysis

• Nonlinear regression analysis with inhibitor vs. normalized response

• Statistical analysis using software such as GraphPad Prism

Using this approach, synthetic Kalata B1 demonstrated IC50 values of 2.6 μM in T-98G cells and 5.4 μM in U-87 MG cells, while natural Kalata B1 showed comparable activity with IC50 values of 5.6 μM in SH-SY5Y cells and 7.9 μM in U-87 MG cells .

What protocols can detect the formation of lipidic toroidal pores by Kalata B1?

Kalata B1 has been shown to form lipidic toroidal pores in membranes . While detailed protocols weren't fully described in the provided research, fluorescent dye leakage assays using model membrane mimetics have been employed to confirm the membrane lytic ability of cyclotides . The relative potency of different cyclotides (such as Kalata B1 and Kalata B2) in causing membrane leakage correlates with their biological activity, supporting membrane disruption as a key mechanism of action .

How might the conditional intein system be optimized for larger-scale production of Kalata B1?

The conditional intein system represents a versatile approach for recombinant cyclotide production with potential for optimization through:

• Engineering enhanced intein precursors with improved efficiency

• Optimizing expression conditions for higher yields

• Developing streamlined purification protocols

• Exploring alternative host organisms for expression

• Investigating continuous processing methods for the cyclization reaction

These optimizations could facilitate larger-scale production while maintaining the precision control offered by the conditional intein system.

What potential exists for engineering Kalata B1 variants with enhanced therapeutic selectivity?

The established structure-function relationships of Kalata B1 provide a foundation for rational design of variants with enhanced therapeutic selectivity. Potential approaches include:

• Modifying surface-exposed residues to alter membrane interaction profiles

• Engineering chimeric cyclotides that combine structural elements from different natural cyclotides

• Incorporating targeting moieties to direct activity toward specific cell types

• Exploring the impact of D-amino acid substitutions on selectivity profiles

The unique stability of the cyclotide scaffold makes it an excellent candidate for such engineering efforts, potentially yielding variants with improved therapeutic indices for applications like targeted cancer therapy.

How might computational modeling assist in predicting structure-activity relationships in Kalata B1 variants?

While not explicitly addressed in the provided research, computational modeling represents a valuable approach for cyclotide engineering. Methods might include:

• Molecular dynamics simulations to predict stability of engineered variants

• Membrane interaction modeling to optimize surface properties

• Structure-based design of variants with enhanced target selectivity

• Prediction of cyclization efficiency based on sequence modifications

Such computational approaches could significantly accelerate the development of novel cyclotide variants with tailored properties for specific research or therapeutic applications.