Methods for displaying cyclic peptides on bacteriophage particles
The present invention relates to methods for displaying cyclic peptides on the surface of bacteriophage particles and collections thereof.
1. A method for displaying monocyclic and polycyclic peptides up to 500 amino acids in length on the surface of a bacteriophage particle comprising the following steps:
(a) providing a host cell harbouring a nucleic acid sequence encoding a precursor cyclic peptide, a coat protein of a bacteriophage particle and a consensus motif which is recognized by a LanB type dehydratase, a LanC type cyclase, a bifunctional LanM type enzyme or a multifunctional LanKC or LanL type enzyme;
(b) causing or allowing the expression of said precursor cyclic peptide, said coat protein and said consensus motif;
(c) enzymatic dehydration of one or more amino acid residues within the precursor cyclic peptide prior to phage assembly;
(d) forming one or more intramolecular bonds by coupling of said one or more dehydrated residues to a cysteine or a lysine, thereby forming a cyclic peptide prior to phage assembly; and
(e) producing bacteriophage particles in said host cell, wherein said bacteriophage particles export and display said cyclic peptide on the surface, wherein said cyclic peptide is attached to the C-terminus of a coat protein of said bacteriophage particles, and wherein said cyclic peptide is selected from monocyclic peptides, polycyclic peptides with 2, 3, 4 and 5 intramolecular bonds, and cyclic peptides up to 500 amino acids in length.
2. The method of claim 1 , wherein the host cell further harbours one or more nucleic acid sequences encoding a post-translationally modifying (PTM) enzyme.
3. The method of claim 1 , wherein the one or more dehydrated amino acid residues are dehydroalanine (Dha) or dehydrobutyrine (Dhb).
4. The method of claim 1 , wherein the intramolecular bond is a thioether- or lysinoalanine-bridge.
5. The method of claim 2 , wherein said post-translationally modifying (PTM) enzyme is a lanthipeptide synthetase.
6. The method of claim 2 , wherein said post-translationally modifying (PTM) enzyme is a LanB type dehydratase, a LanC type cyclase and/or a bifunctional LanM type enzyme or a multifunctional LanKC or LanL type enzyme.