Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A

• Cyclotides exhibit a cyclic polypeptide backbone and have utility in drug discovery.
• The cyclotide MCoTI-II was generated by recombinant expression and purification.
• Engineered Sortase A was used to catalyze backbone cyclization of MCoTI-II.
• Engineered Sortase A is more efficient than wild-type enzyme at cyclizing MCoTI-II.
• Folding but not cyclization influences biological activity and stability of MCoTI-II.

Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.