title: Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A.
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.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Stanger
authors.forename: Till
authors.initials: T
authors.lastname: Maurer
authors.forename: Harini
authors.initials: H
authors.lastname: Kaluarachchi
authors.forename: Mary
authors.initials: M
authors.lastname: Coons
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Franke
authors.forename: Rami N
authors.initials: RN
authors.lastname: Hannoush
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003500:Cyclization; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003553:Cystine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D003553:Cystine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases