IN VITRO PRODUCTION OF CYCLIC PEPTIDES
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.
This invention relates to methods for the production of cyclic peptides in vitro.
Cyclic peptides have long been of interest to the biotechnology and pharmaceutical industries for use as novel medicines. They are considerably more stable compounds than linear peptides and can cross cell membranes more efficiently, which makes them ideal drug molecules (Driggers, E. M. et al. Nat Rev Drug Discov 7, 608-624 (2008))). Cyclic peptides are particularly challenging to produce synthetically. Marine cyanobacteria have been shown to produce cyclic peptide natural products, the cyanobactins (Sivonen et al., 2010, Appl Microbiol Biotechnol, 86, 1213-25; See
Patellamides, members of the cyanobactin superfamily, are produced by Prochloron spp., an obligate, uncultured symbiont of the sea squirt Lissoclinum patella (Schmidt et al., 2005, Proc Natl Acad Sci USA, 102, 7315-20; Long et al. 2005, Chembiochem, 6, 1760-5). These compounds show cytotoxicity (Kohda et al., 1989, Biochem Pharmacol, 38, 4497-500) and the ability to reverse multiple drug resistance in human leukemia cells (Williams and Jacobs, 1993, Cancer Lett, 71, 97-102). Patellamides are cyclic octapeptides containing heterocyclized residues (Ser/Thr, Cys) giving oxazolines and thiazolines, which can be further oxidized to thiazoles (Schmidt et al., 2005, Proc Natl Acad Sci USA, 102, 7315-20).
The Patellamide gene cluster has been identified and the genes patA, D, E, F, and G have been reported to be essential to yield products (Donia et al., 2008, Nat Chem Biol, 4, 341-3; Donia et al., 2006, Nat Chem Biol, 2, 729-35). PatE, the pre-pro-peptide, consists of 37-residue leader sequence (containing a single helix from residues 13-28 {Houssen, W. E. et al. —Chembiochem 11, 1867-1873}), and one, two or three cassettes of eight residues, which are each flanked by N- and C-terminal protease recognition sites and go on to form the final product {Schmidt et al., 2005, Proc Natl Acad Sci USA, 102, 7315-20}.
Several steps in the synthesis of patellamides have been fully characterised. Heterocyclization of specific amino acids must come before N- and C-terminal cleavage, with macrocyclisation being the final step to product. It is still unclear at what stage epimerization, oxidation of thiazolines to thiazoles, and prenylation and/or geranylation occur but epimerisation has been reported to be spontaneous and occurs after macrocyclisation. Oxidation must be last. (Milne, B. F. et al Org Biomol Chem 4, 631-638 (2006)). This application focuses on the heterocyclisation, cassette cleavage and macrocyclization steps.
Heterocyclization is the first step in PatE pre-pro-peptide tailoring and catalyzed by the three-domain protein PatD. PatD contains substrate specificity for the 37 amino acid leader sequence of PatE and heterocyclises cysteine and threonine/serine residues to form thiazolines and oxazolines respectively. This process results in the loss of one water molecule per heterocycle. TruD, a PatD homolog from the trunkamide pathway has been shown to heterocyclize cysteine residues only (McIntosh, J. A. et al (2010). Chembiochem 11(10): 1413-1421).
The N-terminal cleavage of the cassette is catalyzed by PatA, a two-domain protein consisting of an N-terminal subtilisin-like protease domain and a C-terminal domain of unknown function (DUF). The protease domain (PatApr) acts on the cleavage recognition sequence ‘G(L/V)E(A/P)S’, with the first residue of the cassette in the P1′ position. {Lee et al., 2009, J. Am. Chem. Soc., 131, 2122-2124}
The final step of patellamide production is C-terminal cleavage and macrocyclisation. This step is catalysed by PatG, a three-domain protein consisting of an N-terminal oxidase domain, a subtilisin-like protease/macrocyclase domain and a C-terminal DUF. The protease/macrocyclase domain (PatGmac) is responsible for both cleavage of the C-terminus of the cassette and for macrocyclizing the cleaved cassette into a patellamide. {Lee et al. 2009 J. Am. Chem. Soc., 131, 2122-2124} PatGmac recognises the sequence XAYDG, where X is the final residue in the cassette, located in the P1 position. {McIntosh et al., 2010, J Am Chem Soc, 132, 15499-501} It has been reported previously that the final residue of the cassette must be a Pro or heterocycle {McIntosh et al., 2010, J Am Chem Soc, 132, 15499-501}.
Previous in vivo studies of the pathway have shown that cyclic products yields of up to 320 μg/L can be produced (Tianero M D et al. JACS (2012) 418-425).
This invention relates to the development and optimisation of in vitro methods for the production of cyclic peptides using cyanobacterial enzymes, such as patellamide biosynthesis enzymes. This may be useful, for example, for the production of peptidyl molecules, the biosynthesis and screening of candidate therapeutics, and nanotechnology applications.
An aspect of the invention provides an in vitro method of producing a cyclic peptide comprising;
(i) providing a linear peptide substrate; and,
-
- (ii) treating said peptide substrate with an isolated cyanbacterial macrocyclase to produce a cyclic peptide.
Cyclic peptides are circularised peptidyl compounds which include cyclotides and cyanobactins, for example patellamides and telomestatins. Patellamides are cyclic octapeptides produced by Prochloron spp which include patellamide A, B, C and D.
A cyanobacterial macrocyclase is a cyanobacterial enzyme which catalyses the cyclisation of peptide substrates which contain a cyclisation signal.
Suitable cyanobacterial macrocyclases include PatG macrocyclase (AAY21156.1 GI:62910843; residues 492-851 of SEQ ID NO: 1) and TruG (gi|167859101|gb|ACA04494.1) from Prochloron and macrocylases from Anabaena spp, such as ADA00395.1 GI:280987232; ACK37889.1 GI:217316956 and AED99446.1 GI:332002633; Oscillatoria sp, such as GI:300866529 ZP—07111219.1; Microcystis spp such as GI:389832527 CCI23764.1, GI:158934376 CA082089.1, GI:389788443 CCI15902.1, GI:389678154 CCH92964.1, GI:389802072 CCI18832.1, GI:389882395 CCI37144.1, GI:389826370 CCI23111.1; GI:389731219 CCI04703.1, GI:389716328 CCH99432.1, GI:389831597 CCI25524.1 and GI:159027550 CA086920.1; Nostoc spongiaeforme spp, such as TenG (GI:167859092 ACA04486.1); lyngya spp, such as GI:119492374 ZP—01623710.1; Nodularia spp, such as GI:119512474 ZP—01631555.1; Anabaena spp, such as AcyG (GI:280987232 ADA00395.1) Planktothrix spp, such as GI:332002633 AED99446.1, Trichodesmium spp, such as GI:113475997 YP—722058.1; and Arthrospira spp, such as ZP—06384654.1 GI:284054444, GI:284054071 ZP—06384281.1, GI:291571075 BAI93347.1, GI:284054444 ZP 06384654.1, and GI:376002294 ZP—09780130.1. The sequence alignment of Table 4 provides the sequences of other suitable cyanobacterial macrocyclases.
Other suitable cyanobacterial macrocyclases are available in the art (Lee, S. W. et al (2008). Discovery of a widely distributed toxin biosynthetic gene cluster, PNAS 105(15), 5879-5884).
A cyanobacterial macrocyclase may comprise the amino acid sequence of any one of the above reference cyanobacterial macrocyclase sequences or may be a variant thereof. For example, a cyanobacterial macrocyclase may be a PatG macrocyclase which comprises the amino acid sequence of residues 492-851 of SEQ ID NO: 1 or other macrocyclase shown in Table 4 or which comprises an amino acid sequence which is a fragment or variant thereof.
In some embodiments, a PatG macrocyclase may comprise the sequence of SEQ ID NO: 1 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, up to 15, up to 20, up to 30, up to 40, up to 50, or up to 60 residues may be inserted, deleted or substituted. Suitable residues for substitution include R589, K594, K598 and H746.
The position in a cyanobacterial macrocyclase which corresponds to position R589, K594, K598, H746 or other position of the PatG sequence of SEQ ID NO: 1 may be readily determined using routine sequence analysis techniques. The amino acid at this position may be replaced by a different amino acid residue using routine site-directed mutagenesis techniques (see for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al. (2001) Cold Spring Harbor Laboratory Press).
Fragments and variants of a reference sequence are described elsewhere herein. In some embodiments, a cyanobacterial macrocyclase which comprises a sequence which is a variant of one of the above reference sequences may comprise Asp, His and Ser residues at positions equivalent to Asp548, His618 and Ser783 in SEQ ID NO: 1.
A cyanobacterial macrocyclase which comprises a sequence which is a variant of one of the above reference sequences may comprise the residues shown in black in a macrocyclase sequence shown in the alignment of Table 4 in an equivalent position in the variant sequence.
In some embodiments, the cyanobacterial macrocyclase may comprise a modified recognition sequence which recognises a modified cyclisation signal. The recognition sequence in the macrocyclase and the cyclisation signal in the peptide substrate may be modified such that they are complementary and binding between macrocyclase and substrate occurs. For example, one of the macrocyclase and the cyclisation signal may be a positive sequence, such as RRR or KKK, and the other may be a negative sequence, such as DDD or EEE. In some embodiments, the cyanobacterial macrocyclase may comprise the recognition sequence RKK which recognises the cyclisation sequence AYDG.
In some embodiments, the cyanobacterial macrocyclase may comprise a substitution at the residue equivalent to H746 and/or F747 of SEQ ID NO: 1. These residues interact with the Y of the cyclisation signal AYD. For example, substituting F747 to a charged residue in the macrocyclase may allow substitution of Y for residue with opposite charge in the cyclisation signal.
In some embodiments, the cyanobacterial macrocyclase may comprise a substitution at the residue equivalent to K598 of SEQ ID NO: 1. For example, the cyanobacterial macrocyclase may comprise a K598D substitution and may recognise the cyclisation signal AYR.
Modification of the cyanobacterial macrocyclase sequence, for example by a R589, K594, K598 and H746 or other substitution or equivalents, may have improved activity and/or kinetics over the native enzyme sequence. This may be helpful in making the biosynthetic process viable in a reasonable time.
Modification of the cyanobacterial macrocyclase sequence to recognise a modified cyclisation sequence may be required if the target peptide sequence for cyclisation contains an unmodified cyclisation sequence (e.g. XAYD, where X is a heterocycle or Pro).
The peptide substrate may comprise a target peptide and a C terminal cyclisation signal.
The target peptide is the sequence which undergoes cyclisation by the macrocyclase to form the cyclic peptide.
A suitable target peptide may have at least 4, 5, 6, 7 or 8 residues. A suitable target peptide may have up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more residues. For example, a suitable target peptide may have from 4 to 30 residues, preferably 4 to 23 residues, more preferably 6 to 23, 6 to 20 or 6 to 11 residues.
The target peptide sequence may be natural e.g. a natural cyanobactin sequence or a precursor thereof; or a natural cyclotide sequence or a precursor thereof; or the target peptide sequence may be synthetic. For example, the target peptide sequence may be a heterologous sequence which is not normally associated with a cyanobactin cyclisation signal.
The target peptide may include modified amino acids, unmodified amino acids, heterocyclic amino acids, non-heterocyclic amino acids, naturally occurring amino acids and/or non-naturally occurring amino acids.
Methods of the invention also provide the introduction of heterocyclic amino acids into the target peptide sequence using isolated cyanobacterial enzymes, as described below, and optionally the oxidation of the introduced heterocyclic amino acids.
A target peptide sequence may comprise 0, 1, 2, 3, 4, 5, 6, 7, 8 or more heterocyclic amino acids (Shin-ya, K. et al J. Am. Chem. Soc. 2001, 123, 1262-1263).
Preferably, the residue directly N terminal to the cyclisation signal in the target peptide sequence is a heterocyclic amino acid. For example, an amino acid selected from thiazoline (Thn), thiazole (Thz), oxazoline (Oxn), oxazole (Oxz), proline and pseudoproline (*Pro).
In other embodiments, the residue directly N terminal to the cyclisation signal in the target peptide sequence may be an N-methylated amino acid or a moiety with an NH2 and COOH group which allows the target peptide sequence to bend sufficiently for macrocyclisation.
Suitable target peptide sequences include ITACITFC; ITACISFC; ICACITFC; IAACITFC; ITACITYC; ITACITAC; ITA(SeCys)ITF(SeCys); IMACIMAC; IDACIDFC; ITVCITVC; ITAAITFC; VPAPIPFP; VTVCVTVC; VGAGIGFP; ACIMAC; IACIMAC; IITACIMAC; ATACITFC and GVAGIGFP. Other suitable target peptide sequences, for example cyanobactins or other cyclic and macrocyclic peptides, are well-known in the art (see for example Houssen, W. E. & Jaspars, M. Chembiochem 11, 1803-1815 (2010); Sivonen, K., et al (2010) Applied Microbiology, (86) 1213-1225) and/or described elsewhere herein.
Other suitable target peptide sequences include cyclotide sequences, such as GLPVCGETCVGGTCNTPGCTCSWPVCTRN (Kalata B1).
In some embodiments, one or more residues in the target peptide sequence may comprise a reactive functionality which may allow further chemical modification. Suitable residues may contain side chains with side chain linking groups such as NH2, COOH, OH and SH.
The cyclisation signal is located at the C terminal of the peptide substrate, preferably adjacent the target peptide. The cyclisation signal is the recognition site for the cyanobacterial macrocyclase. The sequence of the cyclisation signal in a peptide substrate may depend on the cyanobacterial macrocyclase being used. Typically, a cyclisation signal will comprise the sequence; small residue—bulky residue—acidic residue. Suitable cyclisation signals include AYD, AYE, SYD, AFD and FAG.
In some preferred embodiments, the cyanobacterial macrocyclase is a PatG macrocyclase and the cyclisation signal is AYD.
In some embodiments, the cyclisation signal may be heterologous i.e. not naturally associated with the target peptide sequence.
The cyclisation signal may be a natural cyclisation signal or a synthetic or modified cyclisation signal. A modified cyclisation signal may be recognised by a modified cyanobacterial macrocyclase, as described above.
The linear peptide substrate may be treated with the cyanobacterial macrocyclase under suitable conditions for the cyclisation of peptide.
Suitable conditions would be apparent to those skilled in the art. In some preferred embodiments, conditions may include 500 mM NaCl and/or pH 9. For example, the linear peptide substrate may be treated with the cyanobacterial macrocyclase in 500 mM NaCl and 5% DMSO at pH 8.
The highest temperature tolerated by the macrocylase is generally preferred as this leads to increased reaction rates. The optimal temperature for reaction under a defined set of conditions may be determined experimentally.
In some embodiments, the linear peptide substrate may be immobilised, for example on a solid support, and the cyanobacterial macrocyclase may be free in solution. This may be useful, for example in facilitating purification of the cyclic peptide.
In other embodiments, the linear peptide substrate may be free in solution and the cyanobacterial macrocyclase may be immobilised for example on a solid support, such as a bead. This may be useful, for example in facilitating re-cycling of the macrocyclase.
In some embodiments, a linear peptide substrate may be produced, for example by chemical synthesis or recombinant means as described below, and treated directly with the cyanobacterial macrocyclase. This may be useful in producing cyclic peptides which do not contain heterocycles.
In other embodiments, the linear peptide substrate may be produced from a pro-peptide. For example, the linear peptide substrate may be provided by a method comprising;
-
- (i) providing a linear pro-peptide; and,
- (ii) treating said linear pro-peptide with an isolated protease to produce the linear peptide substrate.
The linear pro-peptide may comprise the linear peptide substrate linked to a pro-sequence, for example an N terminal leader sequence, by a protease recognition site.
In some embodiments, the protease recognition site may be G(L/V)E(A/P)S and the protease may be a cyanobacterial protease, such as a PatA protease. Other suitable protease recognition sites include GLEAS, GVEPS, GVEPP, GVDAS, GVGAS, GAGAS, GAEAS, QVQAQ, QVEAQ, QVQAL, QVTAQ, QVTAH, QVTPH, GPGPS and RVTVQ.
A cyanobacterial protease is an enzyme from a cyanobacterium which cleaves a peptide chain at a protease recognition site.
Suitable cyanobacterial proteases include PatA protease (AAY21150.1 GI:62910837), TruA protease (ACA04487.1 GI:167859094) from Prochloron spp and proteases from Lyngbya sp, such as ZP—01623699.1 GI:119492363; Microcystis spp, such as CA086912.1 GI:159027542; and CA082081.1 GI:158934368; Nostoc spongiaeforme spp, such as TenA (ACA04480.1 GI:167859086); Anabaena spp, such as AcyA (ACK37888.2 GI:280987221), Oscillatoria sp such as ZP—07111214.1 GI:300866524; Trichodesmium spp, such as YP—722055.1 GI:113475994; Nodularia spp, such as ZP—01631559.1 GI:119512478; Cyanothece spp, such as YP—003900371.1 GI:307591572 and YP 002481258.1 GI:220905947; and Arthrospira spp, such as BAI93369.1 GI:291571097. Other suitable cyanobacterial proteases are shown in Table 5.
A cyanobacterial protease may comprise the amino sequence of any one of the above reference cyanobacterial protease sequences or may be a variant thereof. For example, a cyanobacterial protease may be a PatA protease which comprises the amino sequence of SEQ ID NO: 2 or is a variant thereof. Variants of a reference sequence are described elsewhere herein.
In some embodiments, the cyanobacterial protease may comprise a modified sequence which recognises a modified and/or heterologous protease recognition site. The protease sequence and the protease recognition site in the peptide substrate may be modified such that they are complementary and binding occurs.
In more preferred embodiments, the pro-peptide may further comprise a heterologous protease recognition site and the protease may be a heterologous protease.
For example, the heterologous protease recognition site may be a K or R residue and the protease may be trypsin; the heterologous protease site may be Y and the protease may be chymotrypsin; the heterologous protease site may be LVPRGS and the protease may be thrombin; the heterologous protease site may be I(E/D)GR and the protease may be factor Xa; or the heterologous protease site may be ENLYFQ(G/S) or ENLYFQ and the protease may be Tobacco Etch Virus (TEV) protease. Other suitable site specific proteases are well-known in the art and any site specific endoprotease with a residue preference may be used. For example, GluC cuts after E, so replacing K or R in the heterologous protease recognition site with E would allow cleavage by GluC.
Heterologous site-specific proteases, such as TEV protease, trypsin and chymotrypsin are well known in the art and are available from commercial sources.
The cyanobacterial protease recognition site may also be a recognition site for the cyanobacterial heterocyclase. When a heterologous protease recognition site is present, the cyanobacterial protease recognition site may be retained in order to allow the introduction of heterocycles into the target peptide sequence, as described below. For example, a linear pro-peptide may comprise the sequence GLEASK[peptide sequence] or GLEASENLYFQ[peptide sequence].
In embodiments in which heterocycles are not introduced into the target peptide sequence, the pro-peptide may lack a cyanobacterial protease recognition site.
In some embodiments, the linear pro-peptide comprises one, two, three or more peptide substrates linked by protease recognition sites. Treatment of the linear pro-peptide with the protease releases the one, two, three or more linear peptide substrates from the pro-peptide. The releases of two, three or more peptide substrates in the linear pro-peptide may be the same or different.
In some embodiments, the pro-peptide may be immobilised and the protease may be free in solution. This may be useful, for example, in facilitating purification of the peptide substrate, for example before cyclisation.
In other embodiments, the pro-peptide may be free in solution and the protease may be immobilised. This may be useful, for example, in facilitating re-cycling of the protease.
Before cyclisation and optionally proteolysis, the linear peptide substrate or pro-peptide may be treated to heterocyclise amino acid residues in the target peptide sequence. For example, the linear peptide substrate or the linear pro-peptide may be provided by a method comprising;
-
- (i) providing a pre-pro-peptide comprising one or more heterocyclisable amino acids;
- (ii) treating said linear pre-pro-peptide with a cyanobacterial heterocyclase to convert the heterocyclisable amino acids into heterocyclic residues,
- thereby producing the linear peptide substrate or the pro-peptide.
Heterocyclisable amino acids include cysteine, selenocysteine, tellurocysteine, threonine, serine, 2,3-diaminopropanoic acid and synthetic derivatives thereof with additional R groups at the alpha and beta position.
The cyanobacterial heterocyclase may convert the cysteine residues in the linear pre-pro-peptide into thiazolines; threonine/serine residues into oxazolines; selenocysteines into selenazolines; tellurocysteines into tellurazolines and/or aminoalanines into imidazolines.
Heterocyclic amino acids include proline.
A cyanobacterial heterocyclase is an enzyme from a cyanobacterium which converts heterocyclisable residues into heterocycles. A cyanobacterial heterocyclase may recognise an N terminal leader sequence and/or a cyanobacterial protease recognition site, as described herein.
Suitable cyanobacterial heterocyclases include PatD heterocyclase (SEQ ID NO:3; AAY21153.1 GI:6291084) or TruD protease (SEQ ID NO: 4; ACA04490.1 GI:167859097) from Prochloron spp and heterocyclases from Nostoc spongiaeforme spp, such as TenD (ACA04483.1 GI:16785908). Other suitable heterocyclases are shown in Table 6.
In some embodiments, cyanobacterial heterocyclase may be selected depending on the residues in the linear pre-pro-peptide that are to be heterocyclised. For example, PatD may be used to heterocyclise Cys, Thr and Ser residues in the linear pre-pro-peptide and TruD may be used to heterocyclise Cys residues in the linear pre-pro-peptide but not Thr or Ser residues.
A cyanobacterial heterocyclase may comprise the amino sequence of any one of the above reference cyanobacterial heterocyclase sequences or may be a variant thereof. For example, a cyanobacterial heterocyclase may be a PatD or TruD heterocyclase which comprises the amino sequence of SEQ ID NO: 3 or 4 or a variant thereof. Variants of a reference amino acid sequence are described elsewhere herein.
In some embodiments, the pre-pro-peptide may comprise a leader sequence. The leader sequence may at the N or C terminal and is recognised by the heterocyclase. N terminal leader sequences may be removed by the protease after heterocyclisation, as described above.
The choice of leader sequence is dependent on the heterocyclase being employed. Suitable N terminal leader sequences include PatE1-34, or PatE26-34, which are recognised by PatD and TruD heterocylases.
The leader sequence may be heterologous.
In other embodiments, the leader sequence may be absent.
In some embodiments, the cyanobacterial heterocyclase may be modified by replacing the recognition domain with a first member of a binding pair. The leader sequence on the pre-pro-peptide may be replaced by the other member of the binding pair. Suitable binding pairs are well known in the art and include glutathione/glutathione binding protein and biotin/streptavidin. For example, the pre-pro-peptide may comprise an N terminal glutathione and the cyanobacterial heterocyclase may comprise a glutathione binding protein domain.
The pre-pro-peptide for heterocyclisation may further comprise a cyanobacterial protease recognition site as described herein which is recognised by the heterocyclase.
Methods of the production of cyanobacterial heterocyclases are described in more detail below.
The pre-pro-peptide may be treated with the cyanobacterial heterocyclase under suitable conditions to heterocyclise one or more heterocyclisable residues therein. For example, the pre-pro-peptide may be treated with the PatD or TruD heterocyclase in aqueous solution at ambient temperature in the presence of Mg2+ and ATP. The highest temperature tolerated by the heterocyclase is generally preferred as this leads to increased reaction rates. The optimal temperature for reaction under a defined set of conditions may be determined experimentally.
In some embodiments, the pre-pro-peptide may be immobilised on a solid support and the cyanobacterial heterocyclase may be free in solution. In other embodiments, the linear pre-pro-peptide may be free in solution and the cyanobacterial heterocyclase may be immobilised on a solid support.
Heterocyclic residues, such as thiazolines, oxazolines, selenazolines, tellurazolines and imidazolines, in the the pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be subjected to oxidation to oxidise one or more heterocyclic residues in the target peptide sequence.
Thiazoline (Thn) residues in the linear pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be oxidized into thiazoles (Thz); oxazoline residues (Oxn) in the linear pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be oxidized into oxazoles (Oxz); selenazolines (Sen) in the linear pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be oxidized into selenazoles (Sez); tellurazolines (Ten) in the linear pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be oxidized into tellurazoles (Tez) and imidazolines (Imn) in the linear pre-pro-peptide, pro-peptide, linear peptide substrate or cyclic peptide may be oxidized into imidazoles (Imz).
Bacterial, cyanobacterial or other enzymatic oxidases or chemical oxidizing agents may be employed.
In some embodiments, the pre-pro-peptide may be treated with a cyanobacterial or other enzymatic oxidase or chemical oxidizing agent following heterocyclisation. Treatment may occur directly after heterocyclisation to oxidise one or more heterocyclic residues in the target peptide sequence or oxidization may occur at a different stage, for example, the cyclic peptide may be treated with the oxidase or chemical oxidizing agent after macrocyclisation.
A cyanobacterial oxidase is an enzyme from a cyanobacterium which oxidises one or more heterocyclic amino acid residues.
Cyanobacterial oxidases may oxidise all the heterocyclic residues described herein or combinations thereof, for example oxazolines and thiazolines; or only thiazolines.
Suitable cyanobacterial oxidases include PatG oxidase (residues 1 to 491 of SEQ ID NO: 1) from Prochloron spp.
A cyanobacterial oxidase may comprise the amino sequence of any one of the above reference cyanobacterial oxidase sequences or may be a variant thereof. For example, a cyanobacterial oxidase may be a PatG oxidase which comprises the amino sequence of residues 1 to 491 of SEQ ID NO: 1 or a fragment, allele or variant thereof.
In some embodiments, bacterial oxidases may be employed to oxidise one or more heterocyclic amino acid residues. Suitable bacterial oxidases are well known in the art and include BcerB oxidase from the thiazole/oxazole modified microcin cluster (Melby et al J. Am. Chem. Soc, 2012, 134, 5309).
Sequences which are fragments or variants of a reference sequence are described below.
In some embodiments, the pre-pro-peptide may be treated with the cyanobacterial oxidase in the presence of flavin mononucleotide (FMN).
In some embodiments, the linear pre-pro-peptide may be immobilised on a solid support and the cyanobacterial oxidase may be free in solution; or the linear pre-pro-peptide may be free in solution and the cyanobacterial oxidase may be immobilised on a solid support.
Alternatively, following heterocyclisation, the pre-pro-peptide, pro-peptide, peptide substrate or cyclic peptide may be treated with a chemical oxidizing agent, such as MnO2. Treatment with the agent may occur directly after heterocyclisation or at a different stage, for example after macrocyclisation. Suitable oxidation conditions may be determined by routine experimentation. For example, a cyclic peptide may be oxidised using MnO2 in dichloromethane for three days at 28° C. to oxidise heterocycles.
Optionally, methods of the invention may further comprise treating a pre-pro-peptide, pro-peptide or peptide substrate with an epimerase, such that one or more amino acids in the target peptide sequence which are adjacent to a thiazoline are converted into D-epimers.
Alternatively, epimerisation of amino acids in the target peptide sequence which are adjacent to a thiazoline residue may be spontaneous and may not require treatment with an epimerase (Milne, B. F. et al Org Biomol Chem 4, 631-638 (2006)).
The linear pre-propeptide, pro-peptide, peptide substrate and/or cyclic peptide may be linked directly or indirectly to a tag. Tags may be useful in detection and purification and suitable tags are described below.
In some embodiments, a linear peptide or cyclic peptide, for example a macrocyclic peptide, may be produced by a method comprising one, two, three, four or more of the enzymatic steps described above. For example, a method of producing a cyclic peptide as described herein may comprise;
-
- providing a pre-pro-peptide;
- treating said pre-pro-peptide with a cyanobacterial heterocyclase,
- treating said pro-peptide with a protease to produce a linear peptide substrate, and
- treating said peptide with a cyanobacterial macrocyclase to produce a cyclic peptide.
The pro-peptide, peptide substrate or cyclic peptide may be treated with a cyanobacterial oxidase or chemical oxidising agent to oxidise heterocycles in the target peptide sequence.
The methods described above may allow the production of more than 1 mg/L of cyclic peptide. For example, the titre of the cyclic peptide in the reaction solution following cyclisation with the cyanobacterial macrocyclase may be more than 500 mg/L or more than 1 g/L.
In some embodiments, the above methods may be used to produce any one of the cyclic peptides described herein.
Following production of a cyclic peptide using a method described above, the cyclic peptide may be further treated.
The cyclic peptide may be produced in dimeric form and may be reduced to convert the dimeric peptides into monomers. Suitable reducing agents and conditions are well-known in the art and include TCEP, DTT and β-mercaptoethanol.
The cyclic peptide may be prenylated and/or geranylated. For example, the cyclic peptide may be treated with a cyanobacterial prenylase.
Cyanobacterial prenylases transfer farnesyl or geranyl-geranyl isoprenoids to a cyclic peptide or a pre-pro-peptide, pro-peptide or peptide precursor as described herein.
Suitable cyanobacterial prenylases include PatF prenylase (GI: 62910842 AAY21155.1, SEQ ID NO: 5), GI: 167859100 ACA04493.1 (TruF2), and GI: 167859099 ACA04492.1 (TruF1) from Prochloron spp; GI: 159027547 CA086917.1, GI: 158934373 CA082086.1, GI: 389788445 CCI15906.1, GI: 389678155 CCH92965.1 (TenF), GI: 166362791 YP 001655064.1, GI:389831610 CCI25499.1, GI:389826377 CCI23120.1, GI: 389826383 CCI23131.1, GI: 389832530 CCI23767.1, GI:389716343 CCH99420.1, GI:389882386 CCI37135.1, GI:389720299 CCH95988.1, GI:389732896 CCI03253.1, GI:389734240 CCIO2071.1, GI:389801748 CCI19127.1 and GI: 389802082 CCI18842.1 from Microcystis spp; GI:167859091 ACA04485.1 (TenF) from Nostoc spongiaeforme spp; GI:119492371 ZP—01623707.1 from Lyngbya spp; GI:280987227 ADA00390.1 (AcyF) from Anabaena sp; GI:376002283 ZP—09780119.1, GI:284054206 ZP—06384416.1 from Arthrospira sp; GI:332002616 AED99429.1 from Planktothrix spp; GI:300866527 ZP—07111217.1 from Oscillatoria spp.; and GI:220905949 YP 002481260.1 from Cyanothece spp.
A cyanobacterial prenylase may comprise the amino acid sequence of any one of the above reference cyanobacterial prenylase sequences or may be a variant thereof. For example, a cyanobacterial prenylase may be a PatF prenylase which comprises the amino acid sequence of SEQ ID NO: 5 or a fragment, allele or variant thereof.
The cyclic peptide may be subjected to further chemical modification.
Suitable modifications include derivatisation with a heterologous moiety, for example, a moiety containing a natural side group such as OH, NH2, COOH, SH, or an unnatural side group suitable for coupling reactions and click chemistry.
Click-chemistry involves the Cu(I)-catalysed coupling between two components, one containing an azido group and the other a terminal acetylene group, to form a triazole ring. Since azido and alkyne groups are inert to the conditions of other coupling procedures and other functional groups found in peptides are inert to click chemistry conditions, click-chemistry allows the controlled attachment of almost any linker to the cyclic peptide under mild conditions. For example, non-cyclised cysteine residues of the cyclic peptide may be reacted with a bifunctional reagent containing a thiol-specific reactive group at one end (e.g. iodoacetamide, maleimide or phenylthiosulfonate) and an azide or acetylene at the other end. Label groups may be attached to the terminal azide or acetylene using click-chemistry. For example, a second linker with either an acetylene or azide group on one end of a linker and a chelate (for metal isotopes) or leaving group (for halogen labelling) on the other end (Baskin, J. (2007) PNAS 104(43)16793-97) may be employed.
The cyclic peptide may be labelled with a detectable label.
The detectable label may be any molecule, atom, ion or group which is detectable in vivo by a molecular imaging modality. Suitable detectable labels may include metals, radioactive isotopes and radio-opaque agents (e.g. gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compounds), radiolucent agents, contrast agents and fluorescent dyes.
The choice of detectable label depends on the molecular imaging modality which is to be employed. Molecular imaging modalities which may be employed include radiography, fluoroscopy, fluorescence imaging, high resolution ultrasound imaging, bioluminescence imaging, Magnetic Resonance Imaging (MRI), and nuclear imaging, for example scintigraphic techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computerised Tomography (SPECT).
In vivo fluorescence imaging techniques involve the creation of an image using emission and absorbance spectra that are appropriate for the particular fluorescent detectable label used. The image can be visualized by conventional techniques, including Fluorescence imaging techniques may include Fluorescence Reflectance Imaging (FRI), fluorescence molecular tomography (FMT), Hyperspectral 3D fluorescence imaging (Guido Zavattini et al. Phys. Med. Biol. 51:2029, 2006) and diffuse optical spectroscopy (Luker & Luker. J Nucl Med. 49(1):1, 2008).
Suitable fluorescence detectable labels include fluorescein, phycoerythrin, Europium, TruRed, Allophycocyanin (APC), PerCP, Lissamine, Rhodamine, B X-Rhodamine, TRITC, BODIPY-FL, FluorX, Red 613, R-Phycoerythrin (PE), NBD, Lucifer yellow, Cascade Blue, Methoxycoumarin, Aminocoumarin, Texas Red, Hydroxycoumarin, Alexa Fluor™ dyes (Molecular Probes) such as Alexa Fluor™ 350, Alexa Fluor™ 488, Alexa Fluor™ 546, Alexa Fluor™ 568, Alexa Fluor™ 633, Alexa Fluor™ 647, Alexa Fluor™ 660 and Alexa Fluor™ 700, sulfonate cyanine dyes (AP Biotech), such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, IRD41 IRD700 (Li-Cor, Inc.), NIR-1 (Dejindom, Japan), La Jolla Blue (Diatron), DyLight™ 405, 488, 549, 633, 649, 680 and 800 Reactive Dyes (Pierce/Thermo Fisher Scientific Inc) or LI-COR™ dyes, such as IRDye™ (LI-COR™ Biosciences)
Other suitable fluorescent detectable labels include lanthanide ions, such as terbium and europium. Lanthanide ions may be attached to the synaptotagmin polypeptide by means of chelates, as described elsewhere herein.
Other suitable fluorescent detectable labels include quantum dots (e.g. Qdot™, Invitrogen). Techniques for labelling proteins with quantum dots are well-known in the art (Michalet, X. et al. Science 307:538, 2005; Alivisatos, P. Nat Biotechnol 22:47-52, 2004).
Magnetic resonance image-based techniques create images based on the relative relaxation rates of water protons in unique chemical environments. Suitable MRI techniques are described in more detail in Gadian, D. ‘NMR and its applications to living systems’. Oxford Univ. Press, 1995, 2nd edition). Magnetic resonance imaging may include conventional magnetic resonance imaging (MRI), magnetization transfer imaging (MTI), magnetic resonance spectroscopy (MRS), diffusion-weighted imaging (DWI) and functional MR imaging (fMRI) (Rovaris et al. (2001) J Neurol Sci 186 Suppl 1: S3-9; Pomper & Port (2000) Magn Reson Imaging Clin N Am 8: 691-713; Kean & Smith, (1986) Magnetic Resonance Imaging: Principles and Applications, Williams and Wilkins, Baltimore, Md.).
Labels suitable for use as magnetic resonance imaging (MRI) labels may include paramagnetic or superparamagnetic ions, iron oxide particles, and water-soluble contrast agents. Superparamagnetic and paramagnetic ions may include transition, lanthanide and actinide elements such as iron, copper, manganese, chromium, erbium, europium, dysprosium, holmium and gadolinium. Preferred paramagnetic detectable labels include gadolinium.
A cyclic peptide may be attached to an antibody molecule, such as an antibody or antibody fragment or derivative, for example for use in antibody-directed drug therapies. Suitable techniques for the conjugation of cyclic peptides and antibodies are well known in the art.
Cyclic peptides produced as described herein may be useful in therapeutics, nanotechnology applications and in optical/electronic or contractile materials.
An isolated enzyme or other protein exists in a physical milieu distinct from that in which it occurs in nature, or in which it was produced recombinantly. For example, the isolated peptide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. The absolute level of purity is not critical, and those skilled in the art can readily determine appropriate levels of purity according to the use to which the protein is to be put.
A heterologous element is an element which is not associated or linked to the subject feature in its natural environment i.e. association with a heterologous element is artificial and the element is only associated or linked to the subject feature through human intervention.
One or more heterologous amino acids, for example a heterologous peptide or heterologous polypeptide sequence, may be joined or fused to a linear peptide substrate, pro-peptide, pre-pro-peptide, macrocyclase, oxidase, heterocyclase, protease or other protein set out herein. For example a pre-pro-peptide may comprise a pre-pro-peptide as described above linked or fused to one or more heterologous amino acids. The one or more heterologous amino acids may include sequences from a source other than cyanobacteria.
In some embodiments, a linear peptide substrate, pro-peptide, pre-pro-peptide, macrocyclase, oxidase, heterocyclase, protease or other protein set out herein may be expressed as a fusion protein with a purification tag. Preferably the fusion protein comprises a protease recognition site between the enzyme sequence and purification tag. Following expression, the fusion protein may be isolated by affinity chromatography using an immobilised agent which binds to the purification tag.
The purification tag is a heterologous amino acid sequence which forms one member of a specific binding pair. Polypeptides containing the purification tag may be detected, isolated and/or purified through the binding of the other member of the specific binding pair to the polypeptide. In some preferred embodiments, the tag sequence may form an epitope which is bound by an antibody molecule.
Various suitable purification tags are known in the art, including, for example, MRGS(H)6, DYKDDDDK (FLAG™), T7-, S- (KETAAAKFERQHMDS), poly-Arg (R5-6), poly-His (H2-10), poly-Cys (C4) poly-Phe (F11) poly-Asp (D5-16), Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL), Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Lett 283, 298-302), SUMO (Marblestone et al Protein Sci. 2006 January; 15(1): 182-189), Cherry tag (Eurogentec), Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.). Known tag sequences are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533. The TAG sequence may be linked to the target protein through a protease recognition site, for example a TEV protease site, to facilitate removal following purification.
In some preferred embodiments, the purification tag is glutathione-S-transferase. Following expression, a fusion protein comprising the linear peptide substrate, pro-peptide, pre-pro-peptide, macrocyclase, oxidase, heterocyclase, protease or other protein set out herein and glutathione-S-transferase may be isolated by affinity chromatography using immobilised glutathione (or vice versa). The purification of glutathione-S-transferase fusion proteins is well known in the art.
In other preferred embodiments, the purification tag is a Small Ubiquitin-like Modifier (SUMO) tag or a His6-SUMO tag. Following expression, a fusion protein comprising the linear peptide substrate, pro-peptide, pre-pro-peptide, macrocyclase, oxidase, heterocyclase, protease or other protein set out herein and the SUMO or His6-SUMO tag may be isolated by affinity chromatography using immobilised glutathione (or vice versa). The purification of SUMO-tagged fusion proteins is well known in the art.
After isolation, the fusion protein may then be proteolytically cleaved to produce the linear peptide substrate, pro-peptide, pre-pro-peptide, macrocyclase, oxidase, heterocyclase, protease or other protein set out herein.
Linear peptide substrates, pro-peptides and pre-pro-peptides as described herein may be generated wholly or partly by chemical synthesis. For example, peptides and polypeptides may be synthesised using liquid or solid-phase synthesis methods; in solution; or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
Chemical synthesis of peptides and polypeptides is well-known in the art (J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984); M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A Users Manual, ABI Inc., Foster City, Calif.; G. A. Grant, (Ed.) Synthetic Peptides, A User's Guide. W. H. Freeman & Co., New York 1992, E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press 1989 and in G. B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997).
Linear peptide substrates, pro-peptides and pre-pro-peptides as described herein may be generated wholly or partly by recombinant techniques. For example, a nucleic acid encoding a linear peptide substrate, pro-peptide and pre-pro-peptide as described herein may be expressed in a host cell and the expressed polypeptide isolated and/or purified from the cell culture.
Macrocyclases, oxidases, heterocyclases, proteases and other enzymes out above may be generated wholly or partly by recombinant techniques. For example, a nucleic acid encoding the enzyme may be expressed in a host cell and the expressed polypeptide isolated and/or purified from the cell culture. Preferably, enzymes are expressed from nucleic acid which has been codon optimised for expression in E. coli.
Nucleic acid sequences and constructs as described above may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.
Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for expression of recombinant polypeptides in cell culture and their subsequent isolation and purification are known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (March 1997) Humana Press Inc).
In some embodiments, macrocyclases, oxidases, heterocyclases, proteases and other enzymes set out above may be expressed as fusion proteins with a purification tag, as described above.
Macrocyclases, oxidases, heterocyclases, proteases and other enzymes set out above and linear peptide substrates, pro-peptides and pre-pro-peptides may be immobilised on a solid support.
A solid support is an insoluble, non-gelatinous body which presents a surface on which the peptides or proteins can be immobilised. Examples of suitable supports include glass slides, microwells, membranes, or beads. The support may be in particulate or solid form, including for example a plate, a test tube, bead, a ball, filter, fabric, polymer or a membrane. A peptide or protein may, for example, be fixed to an inert polymer, a 96-well plate, other device, apparatus or material. The immobilisation of peptides and proteins to the surface of solid supports is well-known in the art.
As described above, cyanobacterial macrocyclases, oxidases, heterocyclases and proteases may comprise an amino acid sequence which is a variant or fragment of a reference amino acid sequence.
A variant of a reference amino acid sequence may have an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the reference amino acid sequence.
Suitable reference amino acid sequences for cyanbacterial cyanobacterial macrocyclases, oxidases, heterocyclases and proteases are provided above.
Amino acid sequence identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego Calif.). GAP uses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), generally employing default parameters.
Particular amino acid sequence variants may differ from that in a given sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, a variant sequence may comprise the reference sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, up to 15, up to 20, up to 30, up to 40, up to 50 or up to 60 residues may be inserted, deleted or substituted.
A fragment is a truncated protein which contains less than the full-length amino acid sequence but which retains the activity of the full-length protein sequence. A fragment may comprise at least 100 amino acids, at least 200 amino acids or at least 300 contiguous amino acids from the full-length sequence.
The methods described herein may be useful in screening cyclic peptides for biological or other activity.
The linear peptide substrate, linear pre-pro-peptide, and/or linear pro-peptide may be immobilised on a bead. In some embodiments, a reference linear peptide substrate, linear pre-pro-peptide, and/or linear pro-peptide which does not include a cyclisation signal may also be immobilised to the same bead.
The bead may be treated with a cyanobacterial macrocyclase as described herein, such that the linear peptide is cyclised and the cyclic peptide may be released from the bead, while the reference peptide substrate lacking the cyclisation signal remains attached.
The released cyclic peptide may then be isolated and screened for a biological activity.
If the cyclic peptide is found to display a biological activity, the bead from which the cyclic peptide was released may be identified and the reference peptide substrate sequenced or otherwise analysed, to allow characterisation of the bioactive cyclic peptide.
Methods as described herein may also be useful in the production and screening of libraries of cyclic peptides. 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 PatGmac 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.
- (i) providing a diverse population of target peptides attached to beads,
The diverse population of target peptides may be spatially arrayed, for example, in one or more multi-well plates, such that the bead from which the cyclic peptide was released can be identified. For example, each individual well in a multi-well plate may contain a homogenous population of target peptides.
The cyclic peptides which are screened may contain one, two, three or more heterocyclic amino acid residues. For example, step (i) of a screening method described above may further comprise;
-
- treating said target peptides with a cyanobacterial heterocyclase to convert heterocyclisable residues in the of target peptides into cyclic residues and,
- optionally further treating the target peptides with an cyanobacterial oxidase to oxidise cyclic residues therein.
Other aspects of the invention provide a peptide substrate as described herein for use in the production of a cyclic peptide and a population of diverse peptide substrates for use in the production of a cyclic peptide library.
A peptide substrate may comprise a target peptide sequence having an N terminal protease recognition site and a C terminal cyclisation signal.
The protease recognition site and/or the cyclisation signal may be heterologous to the target sequence. Preferably the protease recognition site is a trypsin or chymotrypsin recognition site.
The peptide substrate may further comprise an N terminal leader sequence or an N terminal binding moiety.
In some embodiments, the peptide substrate may be directly or indirectly linked to an N and/or C terminal tag.
In some embodiments, the peptide substrate may be immobilized on a solid support, such as a bead. As described above, a reference copy of the target peptide sequence may also be immobilized on a solid support without a cyclisation signal.
A population may comprise peptide substrates as described above, wherein the target peptide sequence is diverse within the population. For example, one, two, three, four or more, or all positions in the target peptide sequence may display diversity i.e. different members of the population may display a different residue at the position.
Preferably, the residue adjacent the cyclisation signal in the peptides in the population is Pro, heterocycle, a N-Me residue or other artificial residue with the correct conformational properties, as described above.
Suitable linear peptide substrates are described in more detail above.
Other aspects of the invention provide materials, reagents and kits and reagents for use in the production of cyclic peptides and populations thereof and the use of such cyclic peptides, for example in screening methods.
Materials may include individual or combinations of isolated pre-pro-peptides, pro-peptides, peptide substrates and recombinant macrocyclases, proteases, oxidases, and heterocyclases as described above. Reagents may be immobilized on solid supports.
A kit may comprise a peptide substrate or library of substrates as described above. For example, a kit may comprise a multi-well plate;
-
- each individual well containing a homogenous population of target peptides 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,
- the sequences of the target peptides being different in different wells.
A kit may further comprise isolated enzyme preparations for use in the methods described above.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents and database entries which are mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments of the invention which are described. Thus, the features set out above are disclosed for use in the invention in all combinations and permutations.
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described herein.
(a) Model of the acyl-enzyme intermediate with AYDG remaining bound at the active site.
(b) The acyl-enzyme intermediate is in equilibrium with the substrate. In PatGmac the amino terminus of the substrate enters the active site, displacing AYDG and leading to macrocyclization. Mutations that disrupt binding of AYDG lead to linear product, as it is hydrolyzed by water. The role of the His in deprotonating the incoming amino terminus is speculative.
Table 1 shows data collection and refinement statistics (molecular replacement) for PatGmac.
Table 2 shows the relative ion counts of linear cleaved and macrocyclized peptide substrate.
Table 3 shows MS data from the cassette ITFCITAC in the PatE peptide treated with the heterocyclase TruD and macrocyclase PatG. The accurate masses of the molecular ion and fragments shown in this table are consistent with the proposed structure (see
Table 4 shows a sequence alignment of PatGmac with its homologs. Secondary structure elements are shown in red. Active site residues are indicated by yellow stars, cysteines involved in disulfide bonding as green triangles (matching directions represent disulfide pairs), residues blocking the S3 and S4 sites as blue diamonds, lysines forming salt-bridges with the substrate as purple circles and His and Phe residues involved in substrate binding are marked by a magenta box.
Table 5 shows cyanobacterial proteases on public databases.
Table 6 shows cyanobacterial heterocyclases on public databases.
Table 7 shows 1H/13C NMR data in CDCl3 at 600/150 MHz for cyclo-I(MxOxn) V(Thn)I(MeOxn)V(Thn) obtained from in vitro biosynthesis.
Table 8 shows 1H/13C NMR data in CDCl3 at 600/150 MHz for cyclo-ITA(Thn)ITF(Thn) from Lissoclinum patella and obtained from in vitro biosynthesis using the peptide substrate ITACITFC.
EXPERIMENTS Materials and Methods 1. Protein Cloning, Expression and Purification 1.1 HeterocyclasesCodon-optimized full length PatD and TruD were cloned into the pJexpress 411 plasmid (DNA2.0 Inc., USA) with an N-terminal His6-tag, with TruD containing an additional Tobacco Etch Virus (TEV) protease cleavage site. Both enzymes are expressed in Escherichia coli BL21 (DE3) cells grown on auto-induction medium (Terrific broth base containing trace elements) for 48 h at 20° C. Cells are harvested by centrifugation at 4,000×g, 4° C. for 15 min. Pellets are re-suspended in 500 mM NaCl, 20 mM Tris pH 8.0, 20 mM imidazole and 3 mM BME and supplemented with 0.4 mg DNAse g−1 wet cells (Sigma) and complete protease inhibitor tablets (EDTA-free, Roche). Cells are lyzed by passage through a cell disruptor at 30 kPSI or by sonication and the lysates are cleared by centrifugation at 40,000×g, 4° C. for 45 min followed by filtration through 0.4 μm membrane filter. Cleared lysates are applied to a Ni-sepharose FF column (GE Healthcare) pre-washed with lysis buffer and the protein eluted with 250 mM Imidazole. The His6-tag of TruD is removed by addition of 1 mg TEV protease per 10 mg TruD incubated at room temperature for 2 hours and the cleaved protein isolated by passage through a second Ni-sepharose FF column. (Note: TruD still functions efficiently if His6-tag is not removed). Both enzymes are then loaded on to a Superdex 200 gel filtration column (GE Healthcare), pre-equilibrated and run in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP. Peak fractions were pooled and the proteins concentrated to 100 μM for use in in vitro reactions.
1.2 MacrocyclasesPatGmac (PatG residues 492-851) was cloned from genomic DNA (Prochloron sp.) into the pHISTEV vector (Liu, H. & Naismith, J. H 2009) and expressed in Escherichia coli BL21 (DE3) grown on autoinduction medium (Terrific broth base containing trace elements; Studier, F. W., 2005) for 48 h at 20° C.
Cells were harvested by centrifugation at 4,000×g, 20° C. for 15 min and resuspended in lysis buffer (500 mM NaCl, 20 mM Tris pH 8.0, 20 mM Imidazole and 3 mM β-mercaptoethanol (BME)) with the addition of complete EDTA-free protease inhibitor tablets (Roche) and 0.4 mg DNase g−1 wet cells (Sigma). Cells were lysed by passage through a cell disruptor at 30 kPSI (Constant Systems Ltd), or by sonication, and the lysate was cleared by centrifugation at 40,000×g, 4° C. for 45 min followed by filtration through 0.4 μm membrane filter. Cleared lysate was applied to a Ni-NTA (Qiagen) column or a Ni-sepharose FF column (GE Healthcare) pre-washed with lysis buffer and protein eluted with 250 mM imidazole.
In some methods, the protein was then passed over a desalting column (Desalt 16/10, GE Healthcare) in 100 mM NaCl, 20 mM Tris pH 8.0, 20 mM imidazole, 3 mM βME. Tobacco etch virus (TEV) protease was added to the protein at a mass-to-mass ratio of 1:10 and the protein digested for 1 h at 20° C. to remove the His-tag. Digested protein was passed over a second Ni-column and the flow-through loaded onto a monoQ column (GE Healthcare) equilibrated in 100 mM NaCl, 20 mM Tris pH 8.0, 3 mM BME. Protein was eluted from the monoQ column through a linear NaCl gradient, eluting at 350 mM NaCl. Finally, the protein was subjected to size-exclusion chromatography (Superdex™ 75, GE Healthcare) in 150 mM NaCl, 20 mM Tris pH 8.0, 3 mM βME, and concentrated to 60 mg mL−1.
In other methods, the protein was then passed over Superdex 75, GE Healthcare in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP and concentrated to 1 mM.
All PatGmac point mutants were produced using the Phusion® site-directed mutagenesis kit (Finnzymes) following the manufacturer's protocol, while the lid deletion mutants were made with fusion PCR. All mutant proteins were expressed and purified as above.
1.3 Precursor PeptidesVariants of PatE, each encoding only one core peptide instead of two tandem patellamide core peptides, was cloned with a C-terminal His6-tag into pBMS233 for easier analysis of processed products. To enable more efficient N-terminal cleavage, additional residues were in some cases added directly before the core peptide to allow for cleavage by either trypsin (K/R) or TEV (ENLYFQ). The protein was expressed in BL21(DE3) cells grown on auto-induction medium (Terrific broth base containing trace elements) at 37° C. overnight. Cells were harvested by centrifugation at 4,000×g, 20° C., for 15 min and re-suspended in 8 M urea, 500 mM NaCl, 20 mM Tris pH 8.0, 20 mM imidazole and 3 mM BME. Cells were lysed by sonication, and the lysate waas cleared by centrifugation at 40,000×g, 20° C. for 45 min followed by filtration through 5, 0.8 and 0.4 μm membrane filters respectively. Cleared lysate was applied to a Ni-sepharose FF column (GE Healthcare) column prewashed with lysis buffer, and protein was eluted with 250 mM imidazole. DDT is added to the eluted PatE to a final concentration of 10 mM and the solution was incubated at room temperature for 3 hours. PatE is further purified and separated from protein aggregates by size-exclusion chromatography (Superdex 75, GE Healthcare) in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP and concentrated to 1 mM.
2. Heterocyclization ReactionsHetrocyclization reactions contained 100 μM PatE, 5 μM TruD/PatD, 5 mM ATP pH 7, 5 mM mgcl2, 150 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM TCEP. Reactions were incubated at 37° C. with shaking at 200 rpm for 24 h when using TruD and 48 h for PatD. In some cases, the PatE showed a degree of precipitation. In these instances the peptide was recovered from the precipitate by denaturation in 8M urea as above followed by Ni affinity chromatography and size-exclusion. Reactions were monitored by MALDI.
Processed PatE was purified on Superdex 75, GE Healthcare in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP and concentrated.
3. Macrocyclization ReactionsFor macrocyclization reactions comparing final product ratios after substrate depletion, 100 μM peptide (VGAGIGFPAYDG) was incubated with 50 μM enzyme in 150 mM NaCl, 10 mM HEPES pH 8, 1 mM TCEP for 120 h at 37° C. Samples were analyzed by ESI or MALDI MS (LCT, Micromass or 4800 MALDI TOF/TOF Analyser, ABSciex).
For other macrocyclization reactions, 100 μM peptide (e.g. VGAGIGFPAYDG, VGAGIGFPAYRG, or GVAGIGFPAYRG) was incubated with 20 μM enzyme in a range of buffers for 24 h at 37° C. (see
Other macrocyclization reactions contained 100 μM peptide (PatE), 5% DMSO, 350 mM NaCl, 20 μM PatGmac, 150 mM NaCl and 20 mM Bicine pH 8.0 were incubated at 37° C. with shaking at 200 rpm for 4 days and monitored by MS.
4. LC-MS Analysis of ProductsLC-MS was performed using a Phenomenex Sunfire C18 column (4.6 mm×150 mm). Solvent A was H2O containing 0.1% formic acid and solvent B was MeOH containing 0.1% formic acid. Gradient: 0-2 min 10% B; 2-22 min 10% B to 100% B; 22-27 min 100% B; 27-30 min 100% B to 10% B. High resolution mass spectral data were obtained from a Thermo Instruments MS system (LTQ XL/LTQ Orbitrap Discovery) coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela PDA autosampler and Accela Pump). The following conditions were used: capillary voltage 45 V, capillary temperature 320° C., auxiliary gas flow rate 10-20 arbitrary units, sheath gas flow rate 40-50 arbitrary units, spray voltage 4.5 kV, mass range 100-2000 amu (maximum resolution 30000).
5. Crystallization, Data Collection, and Crystallographic AnalysisCrystals of PatGmac were obtained in 19% PEG6000, 0.07 M calcium acetate, 0.1 M Tris pH 9.0. The crystals were cryoprotected in 30% glycerol and flash-cooled in liquid nitrogen. These crystals belonged to space group C2 with cell dimensions a=132.1 Å, b=67.6 Å, c=97.3 Å, β=115.0 °.
Crystals of PatGmac with peptide were obtained from a mixture of PatGmac with peptide (VPAPIPFPAYDG, 1:4 molar ratio) in 1.2 M sodium citrate, 0.1 M sodium cacodylate pH 7.0. There was electron density for a peptide at one active site but the quality of the map was poor. We reasoned this was due to low occupancy of the peptide and therefore soaked the complex crystals overnight in 7.5 mM peptide prior to data collection. These crystals belonged to space group C2 with a=135.6 Å, b=67.3 Å, c=137.9 Å, β=116.8°. Diffraction data of both structures were collected in-house, each on a single crystal at 100 K on a Rigaku 007HFM rotating anode X-ray generator with a Saturn 944 CCD detector and processed with xia2 (Winter, G., 2009).
The structure of PatGmac was solved by molecular replacement with PHASER (Storoni, L. C., McCoy, A. J. & Read, R. J., 2004; McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J., 2005) using the structure of AkP (PDB entry 1DBI) as the search model, followed by automatic rebuilding with Phenix (Adams, P. D. et al., 2004). The structure of PatGmac with peptide was solved by molecular replacement using the PatGmac structure as the search model. Manual rebuilding was performed with COOT (Emsley, P. & Cowtan, K. Coot, 2004) and refinement was performed using REFMAC5 (Murshudov, G. N., Vagin, A. A. & Dodson, E. J., 1997) implemented in the CCP4 program suite (Acta Crystallographica Section D 50, 760-763 (1994). The statistics of data collection and refinement are summarized in Table 1. Molecular graphics figures were generated with the program Pymol (DeLano Scientific, LLC).
6. Synthesis of the Peptide SubstratesFmoc amino acid derivatives, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and Fmoc-Gly-NovaSyn® TGT resin were purchased from Novabiochem®, Merck Biosciences, UK. Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), and piperidine were obtained from Sigma-Aldrich, UK and used without further purification.
The peptides, including VGAGIGFPAYDG, VPAPIPFPAYDG, and GVAGIGFPAYRG, were synthesized manually using the standard Fmoc-based strategy (Cammish, L. E. & Kates, S. A., 2000). Amino acids were sequentially coupled after removal of the Fmoc blocking group at each cycle. Fmoc deprotection steps were carried out with 20% piperidine in DMF (v/v) for 12 min while coupling reactions were performed in DMF using a molar ratio of amino acid:HBTU: DIEA:resin of 5:5:10:1. Reactions were monitored using the Kaiser test.
The peptides were cleaved from the support and deprotected by treatment with a mixture consisting of 95% TFA, 2.5% triisopropylsilane (TIPS), and 2.5% H2O (20 mL of mixture g−1 of peptide resin, 3 h at room temperature). The resin was then filtered and washed with TFA. The combined filtrates were concentrated under reduced pressure. The peptide was precipitated with cold diethyl ether and recovered by centrifugation. The peptide sequence was verified by MSMS analysis.
The peptide VGAGIGFPAYRG was purchased from Peptide Protein Research Ltd.
7. Proteolytic CleavageDifferent proteases were used, including trypsin and TEV protease, depending on the PatE sequence created. We use 4 μg of trypsin per 1 mg of purified processed PatE. The corresponding figure for TEV protease is 1 mg for each 10 mg of PatE. Reactions were incubated at 37° C. with shaking at 200 rpm for up to 4 hours. Reaction products are purified using Superdex 30, GE Healthcare in 150 mM NaCl, 20 mM Bicine pH 8.0. The purified product was concentrated using on Phenomenex® Strata C18-E, 55 μm, 70 Å, 2 g/12 mL Giga SPE tube cartridges. After loading the sample, a washing step with deionised water to get rid of buffer salts was carried out and this was followed by elution step with 5× column volume methanol and 5× column volume of acetonitrile. We also washed the column with 5× volume of 0.1% TFA in acetonitrile. Washings with water or acidified acetonitrile were tested separately by MS for all peptides. Peptides were found to be eluted completely with the organic solvents.
8. Purification of PatellamidesMacrocyclisation reactions were concentrated on Phenomenex® Strata C18-E, 55 μm, 70 Å, 2 g/12 mL Giga SPE tube cartridges following the above procedure. This was followed by final purification of the products using HPLC on C4 ACE column 10×250 mm, 5 μm and using a gradient of acetonitrile in water. Water was dionised standard while methanol and acetonitrile are both LCMS standards. All glassware was soaked with 1.0 molar nitric acid (12 hours) and rinsed with deionised water and air or oven dried. Purification process was monitored using DAD at wavelengths of 210, 220, 230, 240 and 254 nm. Structures of the purified products were confirmed using NMR and MS. NMR data for two compounds obtained was tabulated (Tables 7 and 8). Purified compounds were chemically oxidised using MnO2 in dichloromethane for three days at 28° C.
Results Example 1 Overall Structure of the PatG Macrocyclase DomainThe macrocyclase domain of PatG (PatGmac, residues 492-851) was overexpressed in E. coli BL21 (DE3) cells and purified using established protocols (Liu, H. & Naismith, J. H., 2009) The retention profile from gel filtration indicated that the domain was a monomer.
The protein formed crystals belonging to the space group C2, with two biological monomers in the asymmetric unit. The structure was determined at 2.19 Å resolution by molecular replacement using the subtilisin Bacillus Ak.1 protease (AkP) (PDB entry 1DBI) as a search model. Table 1 shows the data collection and refinement statistics. The refined model (PDB entry 4AKS) includes residues 514-653, 659-685, 694-717, 728-745, 754-822, and 835-851 in chain A, and 515-650, 660-688, and 692-850 in chain B. The missing residues are in loops and at the N-terminus and are presumed to be disordered.
PatGmac has a spherical shape with dimensions of approximately 53 Å×42 Å×48 Å. The protein contains a seven-stranded parallel β-sheet with two a helices on each face, a fold common to all subtilisin-like proteases. However, the conserved metal ion of subtilisin-like proteases is not present in PatGmac as the binding site is destroyed by sequence changes.
PatGmac contains a catalytic triad consisting of Asp548 located at the C-terminus of the β-strand β1, His618 in the middle of α4 and Ser783 at the N-terminus of α7. The carboxyl group of Asp548 is hydrogen bonded to the side-chain of His618 (2.9 Å), which is in turn hydrogen bonded to the side-chain of Ser783 (2.7 Å). PatGmac has an insertion that extends from β2 as a loop, then forms a helix-loop-helix motif and creates an N-terminal extension of α4, the helix that harbors His618. The insertion is found in other macrocyclases but is not conserved in length or sequence.
Example 2 Comparison of Subtilisin-Like Protease AkP and PatGmacThe amino acid sequences of the AkP and PatGmac are 28% identical and pairwise superposition gives a Cα rmsd of 1.23 Å over 145 structurally equivalent residues. The most striking difference is that PatGmac contains a helix-turn-helix insertion between α2 and α4 (A574 to K610) that sits above the active site; we denote this as the macrocyclization insertion. Eight of these residues form a two turn N-terminal extension of α4 when compared to the typical subtilisin structure. This results in the catalytic His being in the middle of this helix rather than at the end. The other 29 residues form a helix-turn-helix motif.
Four cysteines, which are highly conserved in PatG and its homologs (Table 4), make two disulfide bonds: Cys685/724 and Cys823/834. The Cys685/724 disulfide bond in PatGmac is different from that seen in subtilisins. Cys137 of AkP is equivalent to Cys685 of PatGmac and it forms an intraloop disulfide bond with Cys139, making an 11-atom ring that is proposed to rigidify the active site.
In contrast, PatGmac Cys685/724 bridges two loops, one of which connects α4 to α6 adjacent to the active site. As a result Phe684 and Arg686 pack against the side-chain of Met660, completely filling the S4 and S3 substrate binding pockets. Cys823/834 links the ends of the loop that connects α8 to α9 at the C-terminus of the domain and is distant from the active site.
Example 3 PatGmac Substrate ComplexThe VPAPIPFPAYDG peptide was chosen to match the residues equivalent to P8-P4′, the eight-residue cassette and four C-terminal residue macrocyclization signature. The proline residues were chosen to mimic the heterocycles of the natural substrate and the peptide can in fact be macrocyclized by PatGmac (albeit slowly).
The structure of the complex of PatGmacH618A (inactive mutant) was determined at 2.63 Å by molecular replacement using PatGmac native as a search model (Table 1). The difference electron density for bound peptide in the active site of one promoter was unambiguous for PIPFPAYDG (P5 to P4′) and showed that three N-terminal residues (VPA) of the substrate mimic are disordered. The refined model (PDB entry 4AKT) contains residues 514-686, 694-719, 727-747, 754-823, and 833-851 in chain A, and 515-651, 657-688, and 692-851 in chain B.
Residues P5 and P4 of the substrate (Pro and Ile) make no contact with the protein while P3 (Pro) has weak van der Waals interactions with Tyr210. P2 (Phe) also makes limited van der Waals contacts and the side chain sits in a shallow pocket. The Pro of P1 adopts a cis peptide conformation that results in the substrate pointing away from the protein and the side-chain makes van der Waals contacts with His618Ala and Val622. The carbonyl of the P1-P1′ peptide is 4.3 Å from and correctly oriented for nucleophilic attack by the hydroxyl of Ser783. The side-chain of Met784 sits on this face of the carbonyl while the side-chain of the absolutely conserved Asn717 points towards the opposite face in the correct position to stabilize the tetrahedral intermediate. The P1′ Ala Ca and side-chain make only a few hydrophobic interactions, including contacts with Met784 and the protein backbone. It sits in a cavity that appears to be large enough for bulkier residues. The P2′ (Tyr) residue makes extensive contacts with the protein: a n-stacking interaction with the highly conserved Phe747, a hydrogen bond to His746 (conserved as His or Lys in homologs) and a hydrogen bond between the Tyr main-chain oxygen and the nitrogen of Thr780. The side-chain of P3′ (Asp) is oriented towards a large electropositive patch created by Arg589, Lys594, and Lys598. It makes a salt bridge with Lys598 and possibly Lys594, though the side chain of Lys594 is not well ordered. The P4′ Gly residue makes no contact with the protein, although the terminal carboxyl group is close to Lys594. The binding of the peptide is accompanied by changes in PatGmac at Phe684, as the main chain moves 2 Å at the Ca position to avoid a clash with the substrate. The side chains of Met660, Phe684 and Arg686 prevent the binding of substrates that adopt an extended conformation.
The active site where the acyl-enzyme intermediate would be formed is shielded from solvent by the macrocyclization insertion and the AYDG peptide.
During macrocyclization, the acyl-enzyme intermediate is in equilibrium with the substrate. In PatGmac, the amino terminus of the substrate enters the active site, displacing AYDG and leading to macrocyclization. Mutations that disrupt binding of AYDG lead to linear product, as it is hydrolyzed by water. The role of the His in deprotonating the incoming amino terminus is speculative.
Example 4 Biochemical Characterization of MacrocyclizationThe peptide VGAGIGFPAYDG was used as a substrate for PatGmac in biochemical assays (
PatGmac is a slow enzyme; turnover rates reported to date are ˜1 per day (Lee, J., McIntosh, J., Hathaway, B. J. & Schmidt, E. W., 2009; McIntosh, J. A. et al., 2010). Increasing the sodium chloride concentration from 150 mM t. 500 mM gave greater than an order of magnitude improvement in rate. Increasing the pH from 8 to 9, further tripled the rate. Adding DMSO gave a small increase in rate but shifted the optimum pH, thus a buffer containing 500 NaCl and 5% DMSO at pH 8 gave a reaction rate over 50 times greater (
Site directed mutants K594D and K598D as well as two deletion mutants 578-608 the helix-loop-helix insertion motif, PatGmacΔ1) and 578-614 (the helix-loop-helix insertion and the N-terminal extension of α4, PatGmacΔ2) consumed substrate at approximately the rate of native protein (
The substrate VGAGIGFPAYRG has a modified recognition sequence (Asp to Arg); as expected PatGmac wild-type (and K594D and R589D/K594D/K598D) reacted extremely slowly with the substrate giving equal amounts of macrocyclized and linear products. PatGmac K598D produced cyclo[VGAGIGFP] with only 8% linear product, at a rate over an order of magnitude faster than wild-type PatGmac with VGAGIGFPAYDG (
Site-directed mutants S783A and H618A (both catalytic triad) gave no detectable reaction. Mass spectrometry clearly identified an acyl-enzyme intermediate (VGAGIGFP-PatGmac) during turnover (
To further explore macrocyclization, PatE pre-pro-peptide (PatE2) was engineered consisting of the 37-residue N-terminal leader sequence and N- and C-terminal cleavage recognition sites flanking a single cassette (ITACITFC) corresponding to the natural product Patellamide D. In addition, a C-terminal His6-tag was added to aid in the purification process (
Precursor peptide PatE2, PatD and TruD (heterocyclases), PatApr (subtilisin-like protease domain) and PatGmac (subtilisin-like protease/macrocyclase domain) were cloned and expressed in E. coli and purified for use in biochemistry reactions (see materials & methods, above).
Example 5 Purification and Refolding of PatE2PatE2 was cloned into the pBMS vector and expressed in E. coli BL21 (DE3) grown in auto-induction medium for 24 hours at 30° C., driving the protein to inclusion bodies. Cells were harvested by centrifugation at 4,000×g for 15 min at 20° C., re-suspended in urea lysis buffer (8 M urea, 500 mM NaCl, 20 mM Tris pH 8.0, 20 mM Imdiazole and 3 mM β-mercaptoethanol (βME)) and lysed by sonication at 15 microns (SoniPrep 150, MSE). The lysate was cleared by centrifugation at 40,000×g, 20° C. followed by passage through a 0.45 μm filter. The cleared lysate was applied to a His-Select column (GE Healthcare) equilibrated with lysis buffer and protein eluted with 250 mM imidazole. The protein was then supplemented with 10 mM Dithiothreitol (DTT) to induce refolding and subjected to size-exclusion chromatography (Superdex 75, GE Healthcare) in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP. The protein eluted as a single monomer peak with final yields of between 250 to 300 mg/L culture.
Example 6 In Vitro Heterocyclization of PatE2In order to assess heterocyclization of our single cassette PatE, we carried out several in vitro reactions. Incubation of 100 μM PatE2 with 5 μM PatD in 150 mM NaCl, 10 mM HEPES pH 7.4, 1 mM TCEP, 5 mM ATP, 5 mM MgCl2 at 37° C. for 30 minutes results in a loss of 72 amu corresponding to the expected four water losses, indicating that both threonine and both cysteine residues within the cassette were heterocyclized (
Alternatively, incubation of 100 μM PatE2 with 5 μM TruD under the same conditions resulted in the expected loss of 36 amu corresponding to two water losses and confirming that only the cysteine residues were heterocyclized (
Of all the enzymes used the two heterocyclases are by far the most difficult to express and purify (40 mg pure protein/L culture). We therefore wanted to investigate if they can be used in smaller amounts and recycled. When the heterocyclization reaction is incubated at 37° C. overnight the amount of enzyme can be reduced from 1:20 to 1:200 (Enzyme:Substrate) but the reaction time is significantly longer.
Passing the finished reaction over a Superdex 5200 gel filtration column (GE Healthcare) gives three peaks: Enzyme, substrate and ATP/ADP (
N-terminal cleavage of the cassette is mediated by the subtilisin-like protease domain of PatA. The protease domain acts on the recognition site ‘GLEAS’, cleaving between the S and the first residue of the cassette. We have found that turnover of this reaction in vitro is a slow process. In fact, incubation of 100 μM PatE2 (with or without prior heterocyclase treatment) with 20 μM PatApr at 37° C. for 200 hours is required for complete cleavage. The cassette portion is purified from PatApr and cleaved leader sequence by injecting the reaction on to a Superdex S30 column (GE Healthcare), pre-equilibrated in 150 mM NaCl, 20 mM Bicine pH 8.1. PatApr is highly expressed in E. coli with yields of >250 mg purified protein per litre of culture.
Due to the slow nature of PatApr, we re-engineered the PatE2 pre-pro-peptide to contain a lysine residue (PatE2K) between the PatA recognition sequence ‘GLEAS’ and the cassette residues to allow for trypsin cleavage (FIG. 11)(e.g. Xn-GLEASK[cassette]-Xm) To test if this addition affected heterocyclase activity, we incubated 100 μM PatE2K separately with 0.5 μM of both PatD and TruD overnight at 37° C. Expected water losses of four and two respectively were found by MS. The heterocyclized peptides were purified as previously described and cleaved with 1:1000 trypsin at 37° C. for 2 hours. Complete cleavage was confirmed by MS (
The PatE2 pre-pro-peptide also re-engineered to contain a TEV protease signal (ENLYFQ)) between the PatA recognition sequence ‘GLEAS’ and the cassette residues to allow for TEV cleavage (e.g. Xn-GLEASENLYFQ[cassette]-Xm
The final stage in patellamide production is C-terminal cleavage and macrocyclization. This step is catalyzed by the PatGmac domain. In order to macrocyclize our single cassette we incubated 100 μM heterocyclized (with either PatD or TruD) and N-terminally cleaved PatE2/PatE2K with 20 μM PatGmac for 24 hours at 37° C. in 20 mM Bicine pH 8.1, 500 mM NaCl, 5% DMSO to complete the reaction. Completeness of the reaction was confirmed by LCT-ESI MS (
PatE substrates with the core sequence cassettes ITVCITVC (TruD), ITACITFC(TruD, PatD), ITACITYC (TruD, PatD), IMACIMAC (TruD), IDACIDFC(TruD), VTVCVTVC(TruD, PatD), ITA(SeCys)ITF(SeCys) (TruD), ACIMAC(TruD),IACIMAC(TruD), IITACIMAC(TruD), ICACITFC(TruD), IAACITFC(TruD), ITACITAC(TruD),ATACITFC(TruD), ITAAITFC (TruD) and ITACISFC (TruD) were treated with either PatD or TruD heterocyclase as indicated, then subjected to proteolysis with trypsin and macrocyclisation with PatGmac, as described above. The cyclic products cyclo(ITV(Thn)ITV(Thn)), cyclo(ITA(Thn)ITF(Thn)), cyclo(I(MeOxn)A(Thn)I(MeOxn)F(Thn)), cyclo(ITA(Thn)ITY(Thn)), cyclo(I(MeOxn)A(Thn)I(MeOxn)Y(Thn)), cyclo-(IMA(Thn)IMA(Thn)), cyclo-(IDA(Thn)IDF(Thn)), cyclo-(VTV(Thn)VTV(Thn), cyclo-(V(MeOxn)V(Thn)V(MeOxn)V(Thn)), cyclo-(ITA(Sen)ITF(Sen)), cyclo-(A(Thn)IMA(Thn)), cyclo-(IA(Thn)IMA(Thn)), cyclo-(IITA(Thn)IMA(Thn)), cyclo-(I(Thn)A(Thn)ITF(Thn)), cyclo-(IAA(Thn)ITF(Thn)), cyclo-(ITA(Thn)ITA(Thn)), cyclo-(ATA(Thn)ITF(Thn)), cyclo-(ITAAITF(Thn)) and cyclo-(ITA(Thn)ISF(Thn)) were then purified and analysed by NMR and MS.
The production of heterocycle-containing macrocyclic structures was confirmed for all of these peptide substrates.
NMR data for cyclo-(I(MeOxn)V(Thn)I(MeOxn)V(Thn)) (Cmpd 32) produced from substrate peptide ITVCITVC and cyclo-(ITA(Thn)ITF(Thn))(Cmpd 33) produced from substrate peptide ITACITFC were tabulated (Tables 7 and 8). Furthermore, the NMR spectrum from in vitro cyclo-(I(MeOxn)V(Thn)I(MeOxn)V(Thn)) was found to correspond to the NMR spectrum of the natural tetrahydroascidiacyclamide produced by Lissoclinum patella The ability to oxidise heterocycles following macrocyclisation was determined by assessing the conversion of thioazolines to thiazoles. Reduced cyclo-(I(MeOxn)V(Thn)I(MeOxn)V(Thn)) produced from substrate peptide ITVCITVC was subjected to oxidation using MnO2 in dichloromethane for three days at 28° C. The resulting mixture was subjected to silica gel and celite column chromatography followed by HPLC chromatography to yield the oxidized product (
A peptide substrate was engineered with a SUMO-tag (Marblestone et al Protein Sci. 2006 January; 15(1): 182-189) and a cassette sequence that previously showed no soluble expression. SUMO tags are small solubility tags (linked to a His6tag) of total size 13.6 kDa (MBP=42 kDa, GST=30 kDa) which can be used to increase the level of soluble expression of a target protein. SDS-PAGE analysis showed that the peptide substrate was expressed in soluble form and the SUMO tag could be removed from the substrate with TEV protease.
Example 11 Use of a Reduced Leader SequenceIt has previously been reported that the leader sequence of PatE is essential for heterocyclisation. We probed the interaction of 15N-PatE with TruD (titrating until two-fold molar excess of TruD to PatE).
Residues 1-15 undergo no change and thus appear uninvolved in binding to TruD. The remainder of the residue signals are broadened to such an extent that they become invisible, indicating that binding occurs at or after residue 16. The most highly conserved sequence in the leader region of PatE spans residues 26-34. A synthetic peptide with the first 25 residues of PatE (Δ25PatE) deleted is processed as efficiently by TruD as native PatE. Three additional peptides were tested Δ37PatE (has only the five residue protease signature prior to the core peptide), Δ42PatE (first residue is core peptide) and the eight-residue core peptide itself. No reaction is seen with the core peptide alone, and surprisingly both Δ37PatE and Δ42PatE peptides are processed at a rate within an order of magnitude of the native, but only one residue of the core peptide (the terminal cysteine) reacts.
Targeting individual residues within the conserved leader region revealed S30 was unimportant (S30F has wild type activity), but L29 and E31 were important. L29R and E31R both processed more slowly and gave mixtures of one and two heterocycles. Mutations G38I, L39N and A41I (within GLEAS protease signature) had no effect on heterocyclisation, while S42Q was processed at a much slower rate and intriguingly gave a mixture of 0 and 2 heterocycles while S42C was processed like wild-type. The mutation A52D was processed much slower, at the rate of S42Q, and also gave a mixture of 0 and 2 heterocycles. In contrast mutations Y53A and D54R, also within the macrocyclization sequence “AYDG” immediately C-terminal, were both processed.
Two PatE mutants with core peptide sequences ITACITFP (C51P) and ITACITFA (C51A) were analyzed. The internal cysteine in C51P heterocyclised (judged by mass spectrometry) within 60 min at 37° C. (similar to native). The C51A mutant PatE without a five-membered ring at the C-terminus on the other hand reacted much more slowly, requiring 16 h at 37° C. for ˜50% product formation.
Example 12 Dimer Formation from Cys Containing Cyclic PeptideThe MALDI mass spectrum of the novel cyclo[VGICAGFP] macrocyclic peptide (6;
Modification of the peptides at the cysteine residues could not be carried out without reducing the disulfide bond first. Reduction was attempted using several different reducing agents, namely TCEP, DTT, β-mercaptoethanol and TCEP immobilized on resin. Reduction with TCEP and DTT were shown to be the most effective, achieving complete reduction of the dimer at t=1 hr, where the peak at 1509 Da completely disappeared and peaks at 745 Da and 767 Da (corresponding to the protonated and sodiated forms of the monomeric cyclic peptide, respectively) appeared. TCEP immobilized on resin and β-mercaptoethanol resulted in partial reduction.
Example 13 Formation of Cyclotides Using the Engineered PatGmacCyclotides e.g. katala B1 are a family of plant proteins (28-40 amino acids) that have a unique topology, which combines a circular peptide backbone and a tightly knotted disulfide network that forms a CCK (cyclic cysteine knot) motif and makes the more than 80 known cyclotides exceptionally stable. The cyclotides are resistant to thermal unfolding, chemical denaturants and proteolytic degradation. There is a wide interest in making these compounds for wide range of applications.
We tested the ability of the engineered PatGmac to macrocyclise the linear peptide sequence of katala B1, monitored the reaction using MALDI and compared the MS of the synthetic product with that of the purified native Katala B1. The reaction substrates were the oxidised and reduced form of the linear peptide sequence and contain at their C-term the recognition signal of PatG (AYDG). PatGmac was found to cyclise both the reduced and oxidised precursors. The reduced precursor gave no traceable starting material after reaction with the enzyme and the oxidised version being less efficient.
Example 14 Formation of Imidazolines Using PatD or TruDA minimal peptide ITASITFXAYDG (where X is g the unnatural amino acid 2,3-diaminopropanoic acid) was incubated with TruD or PatD as described above The reaction was analysed by MALDI MS and shows a loss of 18 Da consistent with heterocycle formation (formation of imidazoline) for both reactions, although the enzyme TruD was more efficient in this reaction (
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Claims
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 PatE1-34 or PatE26-34 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.
Type: Application
Filed: Jun 28, 2013
Publication Date: Nov 12, 2015
Applicants: The University of the University of Aberdeen (Aberdeen), The University Court of the University of St. Andrews (St. Andrews)
Inventors: Wael Houssen Ibrahim (Aberdeen), Marcel Jaspars (Aberdeen), Margaret Smith (Aberdeen), James Naismith (St. Andrews), Jesko Koehnke (St. Andrews), Andrew Bent (St. Andrews), Nicholas Westwood (St. Andrews)
Application Number: 14/410,939
