Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide–Protein Interactions†

Cyclotides are fascinating natural plant micro-proteins ranging from 28 to 37 amino acid residues long and exhibit various biological actions including anti-microbial, insecticidal, cytotoxic, antiviral (against HIV), protease inhibitory, and hormone-like activities.[1–4] They share a unique head-to-tail circular knotted topology of three disulfide bridges; one disulfide penetrates through a macrocycle formed by the other two disulfides, thereby inter-connecting the peptide backbone to form what is called a cystine knot topology (Fig. 1). This cyclic cystine knot (CCK) framework gives the cyclotides exceptional rigidity[5], resistance to thermal and chemical denaturation, and enzymatic stability against degradation.[4, 6] In fact, some cyclotides have been shown to be orally bioavailable. For example, the first cyclotide to be discovered, kalata B1, was found to be an orally effective uterotonic,[7] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[8–10] All of these features make cyclotides ideal tools for drug development.[11–14]