Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

The Potential of the Cyclotide Scaffold for Drug Development.

Cyclotides are a novel class of micro-proteins (≈30-40 residues long) with a unique topology containing a head-to-tail cyclized backbone structure further stabilized by three disulfide bonds that form a cystine knot. This unique molecular framework makes them exceptionally stable to physical, chemical, and biological degradation compared to linear peptides of similar size. The cyclotides are also highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot, and are orally bioavailable and able to cross cellular membranes to modulate intracellular protein-protein interactions (PPIs), both in vitro and in vivo. These unique properties make them ideal scaffolds for many biotechnological applications, including drug discovery. This review provides an overview of the properties of cyclotides and their potential for the development of novel peptide-based therapeutics. The selective disruption of PPIs still remains a very challenging task, as the interacting surfaces are relatively large and flat. The use of the cell-permeable highly constrained polypeptide molecular frameworks, such as the cyclotide scaffold, has shown great promise, as it provides unique pharmacological properties. The use of molecular techniques, such as epitope grafting, and molecular evolution have shown to be highly effective for the selection of bioactive cyclotides. However, despite successes in employing cyclotides to target PPIs, some of the challenges to move them into the clinic still remain.