Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A |
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Affiliation: | 1. Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA;2. Department of Structural Biology, Genentech, South San Francisco, CA, USA |
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Abstract: | 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. |
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Keywords: | Cyclotide Recombinant expression Sortase Protein engineering MCoTI-II Cystine-knot peptide |
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