Macrocyclisation Tags
OwnerClarissa Melo Czekster, Hannes Ludewig +1inventors
InventorsClarissa Melo Czekster, Hannes Ludewig, James Henderson Naismith
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Abstract
This invention relates to the biosynthetic production of macrocyclic molecules from linear precursor peptides that contain highly truncated C terminal recognition sequences of 10 or fewer residues using prolyl oligopeptidase (POP) macrocyclases. This may be useful for example in the biosynthetic production of macrocyclic molecules. Methods and kits for the production of macrocyclic molecules, as well as libraries and methods of screening, are provided.
Claims39
A method of producing a macrocyclic molecule comprising;
- (i) providing a linear precursor peptide consisting of a core region and a cyclisation tag of 10 or fewer amino acid residues;
- and,
- (ii) reacting the precursor peptide with an isolated prolyl oligopeptidase (POP) macrocyclase to produce a macrocyclic molecule having the core region.
A method according to any one of the preceding claims wherein the core region comprises one or more non-peptidyl linkages.
A method according to any one of the preceding claims wherein the cyclisation tag is located at the C terminus of the precursor peptide and is linked directly to the core region through a peptidyl bond.
A method according to any one of the preceding claims wherein the cyclisation tag consists of 3-10 amino acid
A method according to any one of the preceding claims wherein the precursor peptide is reacted with the POP macrocyclase in the presence of a catalytic peptide.
A method according to any one of the preceding claims wherein the catalytic peptide consists of 6-8 residues from the C terminus of the wild type substrate of the POP macrocyclase.
A method according to any one of the preceding claims wherein the POP macrocyclase is a plant POP macrocyclase.
A precursor peptide comprising a core region and a cyclisation tag of 10 or fewer amino acid residues.
A library of precursor peptides comprising a core region and a cyclisation tag of 10 or fewer amino acid residues, wherein the library comprises a diverse core region and the same cyclisation tag.
A method of screening a macrocyclic peptide library comprising;
- (i) providing a population of precursor peptides, each precursor consisting of a core region and a cyclisation tag having 10 or fewer amino acids, wherein the core region is diverse in the population and the cyclisation tag is the same,
- (ii) treating said population with a prolyl oligopeptidase (POP) macrocyclase to convert the core region into a macrocyclic molecule,
- (iii) screening the macrocyclic molecules for activity,
- (iv) identifying an active macrocyclic molecule.
A method of screening a macrocyclic peptide library comprising;
- (i) providing a diverse population of core regions attached to beads, each bead having a first and a second copy of the core regions attached thereto, wherein the first copy but not the second copy is attached to the bead via a cyclisation tag having 10 or fewer amino acids,
- (ii) treating said beads with a prolyl oligopeptidase (POP) macrocyclase to convert the first copy of the core region into a macrocyclic molecule and release the macrocyclic molecules from the beads,
- (iii) screening the macrocyclic molecules for activity,
- (iv) identifying an active macrocyclic molecule
- (v) identifying the bead from which the macrocyclic molecule was released, and
- (vi) sequencing the second copy of the core region attached to the bead.
A kit for use in producing a macrocyclic molecule comprising;
- (i) a precursor peptide comprising a core region and a cyclisation tag of 10 or fewer amino acid residues, and
- (ii) an isolated prolyl oligopeptidase (POP) macrocyclase.
Dependencies are inferred from the claim text.