In vitro production of cyclic peptides

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 cyclisation, residues in the linear peptide substrates may be heterocyclised using isolated cyanbacterial heterocyclases, 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.

1 . A method of producing a cyclic peptide comprising;
 (iii) providing a linear peptide substrate; and,   (iv) treating said peptide substrate with an isolated cyanobacterial macrocyclase to produce a cyclic peptide.   
     
     
         2 . A method according to  claim 1  wherein the linear peptide substrate comprise a target peptide and a C terminal cyclisation signal. 
     
     
         3 . A method according to  claim 2  wherein the target peptide consists of at least 6 residues. 
     
     
         4 . A method according to  claim 2  or  claim 3  wherein the residue in the target peptide adjacent the cyclisation signal is proline, pseudoproline, a heterocyclic residue, or an N-Me residue. 
     
     
         5 . A method according to any one of  claims 2  to  4  wherein the cyclisation signal comprises AYD. 
     
     
         6 . A method according to any one of  claims 1  to  5  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). 
     
     
         7 . A method according to any one of  claims 1  to  6  wherein the cyanobacterial macrocyclase comprises Asp, His and Ser residues at positions equivalent to Asp548, His618 and Ser783 of PatG. 
     
     
         8 . A method according to any one of  claims 1  to  7  wherein the Cyanobacterial macrocyclase comprises the amino sequence of residues 492-851 of PatG (SEQ ID NO:1). 
     
     
         9 . A method according to any one of  claims 1  to  8  wherein the cyanobacterial macrocyclase comprises a substitution at one or more residue equivalent to R589, K594, K598 and H746 of PatG (SEQ ID NO:1), and the linear peptide substrate comprises a modified cyclisation signal. 
     
     
         10 . A method according to any one of  claims 1  to  9  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. 
     
     
         11 . A method according to any one of  claims 1  to  10  wherein one of the linear peptide substrate and the cyanobacterial macrocyclase is immobilised on a solid support. 
     
     
         12 . A method according to any one of  claims 1  to  11  wherein the linear peptide substrate is treated with the cyanobacterial macrocyclase in 500 mM NaCl and/or pH 9. 
     
     
         13 . A method according to any one of  claims 1  to  12  wherein the linear peptide substrate is provided by a method comprising;
 (iii) providing a pro-peptide; and, 
 (iv) treating said pro-peptide with an isolated protease to produce the linear peptide substrate. 
 
     
     
         14 . A method according to  claim 13  wherein the linear pro-peptide comprises the peptide substrate linked to a pro-sequence by a protease recognition site. 
     
     
         15 . A method according to  claim 13  wherein the linear pro-peptide comprises one, two, three or more peptide substrates linked by protease recognition sites. 
     
     
         16 . A method according to any one of  claims 13  to  15  wherein the protease recognition site is G(L/V)E(A/P)S and the protease is a cyanobacterial protease. 
     
     
         17 . A method according to any one of  claims 13  to  15  wherein the protease recognition site is a heterologous protease recognition site and the protease is a heterologous protease. 
     
     
         18 . A method according to  claim 17  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)) and the protease is Tobacco Etch Virus (TEV) protease. 
 
     
     
         19 . A method according to any one of  claims 13  to  18  wherein one of the pro-peptide and the protease is immobilised on a solid support. 
     
     
         20 . A method according to any one of  claims 1  to  19  wherein the linear peptide substrate or the pro-peptide is provided by a method comprising;
 (iii) providing a pre-pro-peptide comprising one or more heterocyclisable amino acids; 
 (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 or the pro-peptide. 
 
 
     
     
         21 . A method according to  claim 20  wherein the PatD or Tru D heterocyclase converts cysteine residues in the pre-pro-peptide into thiazolines. 
     
     
         22 . A method according to  claim 20  or  21  wherein the PatD heterocyclase converts threonine or serine residues in the pre-pro-peptide into oxazolines. 
     
     
         23 . A method according to any one of  claims 20  to  22  wherein the PatD heterocyclase converts selenocysteines in the pre-pro-peptide into selenazolines. 
     
     
         24 . A method according to any one of  claims 20  to  23  wherein the PatD heterocyclase converts the aminoalanines in the pre-pro-peptide into imidazolines. 
     
     
         25 . A method according to any one of  claims 20  to  24  wherein the pre-pro-peptide comprises an N terminal leader sequence. 
     
     
         26 . A method according to  claim 25  wherein the pre-pro-peptide comprises the PatE 1-34  or PatE 26-34  leader sequence. 
     
     
         27 . A method according to any one of  claims 20  to  26  wherein the pre-pro-peptide is treated with the PatD heterocyclase in aqueous solution at ambient temperature. 
     
     
         28 . A method according to any one of  claims 20  to  27  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). 
     
     
         29 . A method according to  claim 28  wherein the PatD heterocyclase comprises the amino sequence of PatD (SEQ ID NO:3) or TruD (SEQ ID NO:4). 
     
     
         30 . A method according to any one of  claims 20  to  29  wherein the method comprises treating the linear peptide substrate, pro-peptide or cyclic peptide to oxidise the heterocyclic residues. 
     
     
         31 . A method according to  claim 30  wherein the heterocyclic residues are oxidised with a chemical oxidising agent. 
     
     
         32 . A method according to  claim 30  wherein the heterocyclic residues are oxidised by treatment with an oxidase enzyme. 
     
     
         33 . A method according to  claim 32  wherein the heterocyclic residues are oxidised by treatment with a cyanobacterial oxidase. 
     
     
         34 . A method according to  claim 33  wherein the cyanobacterial oxidase comprises an amino sequence having at least 60% sequence identity to residues 1 to 491 of PatG (SEQ ID NO:1). 
     
     
         35 . A method according  claim 34  wherein the PatG oxidase comprises the amino sequence of residues 1 to 491 of PatG (SEQ ID NO:1). 
     
     
         36 . A method according to any one of  claims 20  to  35  wherein the cyanobacterial heterocyclase and/or the cyanobacterial oxidase are immobilised on a solid support. 
     
     
         37 . A method according to any one of  claims 1  to  36  wherein the pre-propeptide, the pro-peptide and/or the linear peptide substrate are immobilised on a solid support. 
     
     
         38 . A method according to any one of  claims 1  to  37  wherein the pre-propeptide, pro-peptide and/or linear peptide substrate are linked directly or indirectly to a tag. 
     
     
         39 . A method according to any one of  claims 1  to  38  wherein the cyclic peptide is treated with a cyanobacterial prenylase to produce a prenylated or geranylated cyclic peptide. 
     
     
         40 . A method according to any one of  claims 1  to  39  wherein pre-propeptide, pro-peptide, linear peptide substrate and/or cyclic peptide is subjected to further chemical modification. 
     
     
         41 . A method according to any one of  claims 1  to  40  wherein the cyclic peptide is labelled with a detectable label. 
     
     
         42 . A method according to any one of  claims 1  to  41  wherein the linear peptide substrate, pre-pro-peptide, and/or pro-peptide are immobilised on a bead. 
     
     
         43 . A method according to  claim 42  wherein a reference copy of said linear peptide substrate, pre-pro-peptide, and/or pro-peptide is additionally immobilised to said bead, said reference copy lacking a cyclisation signal. 
     
     
         44 . A method according to  claim 43  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. 
     
     
         45 . A method according to  claim 44  comprising isolating and screening said cyclic peptide to identify a biological activity 
     
     
         46 . A method according to  claim 45  comprising identify the bead which released the cyclic peptide and sequencing the reference copy immobilised on said bead. 
     
     
         47 . A method of screening a cyclic peptide library may comprise;
 (i) providing a diverse population of target peptides attached to beads, each bead having a first and a second copy of the target peptide attached thereto, wherein the first copy but not the second copy is attached to the bead via a cyclisation signal,   (ii) treating said beads with a cyanobacterial macrocyclase to convert the first copy of the target peptide into a cyclic peptide and release the cyclic peptides from the beads,   (iii) screening the cyclic peptides for activity,   (iv) identifying an active cyclic peptide   (v) identifying the bead from which the cyclic peptide was released, and   (vi) sequencing the second copy of the target peptide attached to the bead.   
     
     
         48 . A method according to  claim 47  wherein the population of target peptides is spatially arrayed, such that the bead from which the cyclic peptide was released can be identified. 
     
     
         49 . A method according to  claim 47  or  48  wherein step (i) of the method further comprises treating said target peptides with a cyanobacterial heterocyclase to convert one or more heterocyclizable residues in the target peptides into heterocyclic residues. 
     
     
         50 . A method according to  claim 49  wherein step (i) of the method further comprises treating said target peptides or said cyclic peptides with a cyanobacterial or bacterial oxidase to oxidise heterocyclic cyclic residues therein.