In vitro production of cyclic peptides

Patent metadata

Field	Value
Publication number	US-10494657-B2
Application number	US-201715683266-A
Country	US
Kind code	B2
Filing date	Aug 22, 2017
Priority date	Jun 29, 2012
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

This invention relates to the in vitro production of cyclic peptides using cyanobacterial enzymes, such as patellamide biosynthesis enzymes. Linear peptide substrates are cyclized using an isolated cyanbacterial macrocyclase, such as PatG from Prochloron spp. Before cyclization, residues in the linear peptide substrates may be heterocyclized using isolated cyanbacterial heterocyclasses, such as PatD or TruD heterocyclase. Methods of the invention may be useful, for example, for the production of cyclic peptidyl molecules, including cyclotides, such as katalas, and cyanobactins, such as patellamides and telomestatins, for example for use in the development of therapeutics.

First claim

The invention claimed is:

1. A method of producing a cyclic peptide comprising:
(i) providing a linear peptide substrate comprising a target peptide having 6 to 11 residues and a C terminal cyclisation signal consisting of AND, AYE, SYD, AFD, FAG or AYR; and,
(ii) treating said linear peptide substrate with an isolated cyanobacterial macrocyclase to produce a cyclic peptide, wherein the residue in the target peptide adjacent the cyclisation signal is pseudoproline, a thiazoline, a thiazole, an oxazoline, or an oxazole.

2. The method according to claim 1 , wherein the cyanobacterial macrocyclase comprises an amino acid sequence having at least 60% sequence identity to the amino sequence of residues 492-851 of PatG (SEQ ID NO:1) or an amino sequence selected from the group consisting of SEQ ID NOs: 7 to 19.

3. The method according to claim 1 , wherein the cyanobacterial macrocyclase comprises one or more residues for substitution that includes R589, K594, K598 or H746 of PatG (SEQ ID NO: 1), and the linear peptide substrate comprises a modified cyclisation signal.

4. The method according to claim 1 , wherein the Cyanobacterial macrocyclase comprises a K598D substitution at the residue equivalent to K598 of PatG and the linear peptide substrate comprises the cyclisation signal AYR.

5. The method according to claim 1 , wherein the linear peptide substrate is treated with the cyanobacterial macrocyclase in 500 mM NaCl and/or pH 9.

6. The method according to claim 1 , wherein the linear peptide substrate is provided by a method comprising;
(i) providing a pro-peptide comprising a peptide substrate linked to a pro-sequence by a protease recognition site; and,
(ii) treating said pro-peptide with an isolated protease to produce the linear peptide substrate.

7. The method according to claim 6 , wherein the protease recognition site is a heterologous protease recognition site and the protease is a heterologous protease.

8. The method according to claim 7 , wherein the heterologous protease recognition site is a K residue and the heterologous protease is trypsin; the heterologous protease site is Y and the protease is chymotrypsin; or the heterologous protease site is ENLYFQ(G/S) (SEQ ID NO: 57) and the protease is Tobacco Etch Virus (TEV) protease.

9. The method according to claim 1 , wherein the linear peptide substrate is provided by a method comprising;
(iii) providing a pre-pro-peptide comprising one or more heterocyclisable amino acids; and
(iv) treating said pre-pro-peptide with a PatD or TruD heterocyclase to convert the heterocyclisable amino acids into heterocyclic residues,
thereby producing the linear peptide substrate.

10. The method according to claim 9 , wherein the PatD heterocyclase comprises an amino acid sequence having at least 60% sequence identity to PatD (SEQ ID NO:3) or TruD (SEQ ID NO:4).

11. The method according to claim 9 , wherein the method comprises treating the linear peptide substrate or the cyclic peptide to oxidise the heterocyclic residues.

12. The method according to claim 1 , wherein the cyclic peptide is treated with a cyanobacterial prenylase to produce a prenylated or geranylated cyclic peptide.

13. The method according to claim 1 , wherein the cyclic peptide is labelled with a detectable label.

14. The method according to claim 1 , wherein the linear peptide substrate is immobilised on a bead.

15. The method according to claim 14 , wherein a reference copy of said linear peptide substrate is additionally immobilised to said bead, said reference copy lacking a cyclisation signal.

16. The method according to claim 15 , wherein the cyclic peptide is released from the bead following said treatment with the cyanobacterial macrocyclase and the reference copy remains immobilised to the bead.

17. The method according to claim 16 , further comprising isolating and screening said cyclic peptide to identify a biological activity.

18. The method according to claim 17 , further comprising identify the bead which released the cyclic peptide and sequencing the reference copy immobilised on said bead.

19. The method according to claim 1 , wherein the linear peptide substrate is provided by a method comprising;
(i) providing a pro-peptide comprising a peptide substrate linked to a pro-sequence by a heterologous protease recognition site; and,
(ii) treating said pro-peptide with an isolated heterologous protease to produce the linear peptide substrate,
wherein the heterologous protease recognition site is a K residue and the heterologous protease is trypsin; or the heterologous protease site is Y and the protease is chymotrypsin.

20. The method according to claim 6 , wherein the pro-peptide is provided by a method comprising;
(i) providing a pre-pro-peptide comprising one or more heterocyclisable amino acids; and
(ii) treating said pre-pro-peptide with a PatD or TruD heterocyclase to convert the heterocyclisable amino acids into heterocyclic residues,
thereby producing the pro-peptide.

21. The method according to claim 6 , wherein the method comprises treating the pro-peptide to oxidise the heterocyclic residues.

Assignees

• The Univ Court Of The Univ Of Aberdeen
• The Univ Court Of The Univ Of St Andrews
• Univ Oxford Innovation Ltd

Inventors

• Ibrahim Wael Houssen
• Jaspars Marcel
• Smith Margaret
• Naismith James
• Koehnke Jesko
• Bent Andrew
• Westwood Nicholas

Classifications

• G01N33/6818

  Sequencing of polypeptides · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12Q1/527

  involving lyase · CPC title

• C07K7/64 Primary

  Cyclic peptides containing only normal peptide links · CPC title