WO2017049362A1 - Generation of peptides - Google Patents
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- WO2017049362A1 WO2017049362A1 PCT/AU2016/050897 AU2016050897W WO2017049362A1 WO 2017049362 A1 WO2017049362 A1 WO 2017049362A1 AU 2016050897 W AU2016050897 W AU 2016050897W WO 2017049362 A1 WO2017049362 A1 WO 2017049362A1
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- polypeptide precursor
- amino acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22034—Legumain (3.4.22.34), i.e. asparaginyl endopeptidase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
Definitions
- the present disclosure relates generally to generation of a recombinant enzyme with cyclization activity and its use for generating cyclic peptides as well as linear peptide conjugates.
- proteases are abundant throughout nature and are essential for a wide range of cellular processes. They typically serve to hydrolyze polypeptide chains, resulting in either degradation of the target sequence or maturation to a biologically active form. Less frequently, proteases can act as ligases to link distinct polypeptides, producing new or alternately spliced variants. This unusual function has been reported for processes such as the maturation of the lectin, Concanavalin A (Sheldon et al. (1996) Biochem. J. 320:865- 870), peptide presentation by major histocompatibility complex class I molecules (Hanada et al.
- cyclotides e.g. the prototypical cyclotide kalata B l [kB l]
- PawS-derived trypsin inhibitors e.g. sunflower trypsin inhibitor (SFTI)
- knottin trypsin inhibitors e.g. Momordica cochinchinensis trypsin inhibitor (MCoTI-II)
- MCoTI-II Momordica cochinchinensis trypsin inhibitor
- orbitides e.g. segetalins
- Cyclotides were first identified in the African plant Oldenlandia affinis and exhibit insecticidal, nematocidal and moUuscicidal activity against agricultural pests (Jennings et al. (2001) Proc. Natl. Acad. Sci. U. S. A. 98: 10614-10619; Plan et al. (2008) J. Agric. Food Chem. 56:5237-5241; Colgrave et al. (2008) Biochemistry 47:5581-5589; Colgrave et al. (2009) Acta Trop. 109: 163-166). Other reported activities include neurotensin antagonism (Witherup et al. (1994) J. Nat.
- Cyclotides are characterized by a cystine knot motif that, together with backbone cyclization, confers exceptional stability. This has generated much interest in the cyclotide framework as a pharmaceutical scaffold; a potential heightened by the successful grafting of bioactive sequences into both Mobius and trypsin inhibitor cyclotides (Poth et al. (2013) Biopolymers 00:480-491). Backbone cyclization can also increase the stability and facilitate the oral administration route for bioactive linear peptides, suggesting that this modification will find broad application (Clark et al. (2005) Proc. Natl. Acad. Sci. United States Am. 102: 13767-13772; Clark et al. (2010) Angew. Chem. Int. Ed.
- Cyclotides are produced from precursor molecules in which the cyclotide sequence is typically flanked by N- and C-terminal propeptides.
- the first processing event is the removal of the N-terminal propeptide, producing a linear precursor that remains linked to the C-terminal prodomain (Gillon et al. (2008) supra).
- the final maturation step involves enzymatic cleavage of this C-terminal region and subsequent ligation of the free C- and N- termini.
- only four native cyclases have been identified to date (Barber et al. (2013) supra; Nguyen et al. (2014) supra; Luo et al. (2014) supra; Lee et al. (2009) supra; Gillon et al. (2008) supra).
- serine protease PatG which is responsible for maturation of the bacterial cyanobactins (Lee et al. (2009) supra).
- the serine protease PCY1 reportedly facilitates cyclization of the segetalins, cyclic peptides from the Caryophyllaceae (Barber et al. (2013) supra).
- VPEs vacuolar processing enzymes
- AEPs asparaginyl endopeptidases
- Butelase- 1 was isolated from the cyclotide producing plant Clitoria ternatea and shown to cyclize a modified precursor of kB 1 from O. qffinis, confirming the ability of this group of enzymes to mediate cyclization in vitro (Nguyen et al. (2014) supra) provided that the appropriate recognition sequences are added to the ends of the polypeptide precursor to be cyclized.
- recombinant butelase- 1 from E. coli was only expressed in insoluble form and thus unable to mediate cyclization. Only one AEP with any cyclizing ability has been produced recombinantly, and this was highly inefficient, producing mainly hydrolyzed substrate (Bernath-Levin et al. (2015) supra).
- the present disclosure teaches the production of a functional recombinant asparaginyl endopeptidase (AEP) and its use in an efficient method for producing a cyclic peptide or linear peptide conjugate.
- AEP asparaginyl endopeptidase
- cyclic peptide includes but is not limited to a cyclotide.
- the cyclic peptide may be naturally cyclical or may be artificially cyclized to confer, for example, added stability, efficacy or utility.
- a linear peptide conjugate is the ligation of two or more peptides together in linear sequence.
- the term "peptide” is not to exclude a polypeptide or protein.
- peptide is used to avoid any doubt, the present invention covers a cyclic peptide, cyclic polypeptide and cyclic protein as well as a linear peptide, linear polypeptide or linear protein. All encompassed by the term “cyclic peptide” or “linear peptide”.
- the cyclic peptide or linear peptide can be used in a variety of applications relevant to human and non-human animals and plants. Included are agricultural applications such as the generation of topical agents for treatment of infection or infestation by a pathogen and pharmacological applications such as the treatment of cancer, cardiovascular disease, infectious disease, immune diseases and pain. Therapeutic agents may be delivered topically or systemically.
- both naturally cyclic peptides in linear form and naturally linear peptides can be subject to cyclization as well as linear polypeptide precursors comprising non-naturally occurring amino acids and/or modified side chains or modified cross-linkage bonds.
- the cyclization of a naturally linear peptide can lead inter alia to a longer half life and/or increased stability and/or the ability to be orally administered.
- the cyclization process may be conducted in various ways and can employ prokaryotic or eukaryotic organisms and can act on a polypeptide precursor containing a non-naturally occurring amino acid residue or other modification.
- an asparaginyl endopeptidase (AEP) with cyclizing ability is employed to cyclize a linear polypeptide precursor or ligate together peptides including polypeptides and proteins.
- AEP asparaginyl endopeptidase
- polypeptide includes a "protein”.
- the polypeptide precursor includes a precursor to a naturally cyclic peptide as well as a polypeptide which is naturally linear and is converted into a cyclic peptide.
- the linear polypeptide precursor comprises a C-terminal AEP processing site.
- the C-terminal processing site is an amino acid sequence defined as comprising P3 to PI prior to the actual cleavage site and comprising ⁇ to P3' after the cleavage site towards the C-terminal end.
- P3 to PI and PI to P3 have the amino acid sequence:
- X is an amino acid residue and:
- X 2 is optional or is any amino acid
- X 3 is optional or is any amino acid
- X 4 is N or D
- X 5 is G or S
- X 6 is L or A or I
- X 7 is optional or any amino acid.
- Xi through X 6 comprise the amino acid sequence:
- the N-terminal end of the linear polypeptide precursor may contain no specific AEP processing site or may contain a processing site defined by any one of PI through P3 wherein PI to P3 is defined by:
- X9 is optional and any amino acid or G, Q, K, V or L;
- X 10 is optional or any amino acid or L, F or I or an hydrophobic amino acid residue
- Xii is optional and any amino acid.
- X9 through Xn comprise the amino acid sequence: GLX 11
- the AEP processing site comprises N- and C-terminal end sequences comprising the sequence:
- the C-terminal processing site comprises P4 to PI and ⁇ to P4' wherein PI to P4 and ⁇ to P4' comprise XiX 2 X 3 X 4 XsX 6 X 7 X 8 wherein X 2 to X 7 are as defined above and Xi is optional or any amino acid and X 8 is optional or any amino acid.
- the AEP is produced in the cell and isolated before it is used in vitro with a linear polypeptide precursor to be cyclized.
- the linear polypeptide precursor may also be produced in the cell then separated or otherwise isolated from the cell and cyclized in vitro using the recombinant AEP.
- a polypeptide precursor produced by synthesis, including polypeptides with non-naturally occurring amino acids or a recombinant polypeptide with post-translational modification can also be cyclized in vitro using a recombinant AEP.
- the AEP and polypeptide precursor may also be co- expressed in a compartment of a prokaryotic cell such as but not limited to the periplasmic space. In which case, the resulting cyclic peptide is isolated from the cell.
- a similar protocol is adapted when a eukaryotic organism is employed, such as a yeast (e.g. Pichia sp., Saccharomyces sp. or Kluyveromyces sp.). Genetic material encoding AEP is expressed enabling generation of cyclic peptides in vitro from a precursor polypeptide or in vivo if both the AEP and polypeptide are co-expressed. In either event, the resulting cyclic peptide is subject to isolation and purification from a vacuole or other cellular compartment in the eukaryotic cell or from the reaction vessel.
- yeast e.g. Pichia sp., Saccharomyces sp. or Kluyveromyces sp.
- the AEP and polypeptide precursor are produced in separate eukaryotic cells or in different compartments within the same cell, extracted and then co-incubated in vitro to generate the cyclic peptide.
- the AEP or polypeptide precursor is produced in the eukaryotic cell; the other component is supplied from a different source and the two are then incubated in vitro to generate a cyclic peptide.
- peptide includes a polypeptide or protein as well as a peptide.
- a method for producing a cyclic peptide comprising introducing into the prokaryotic or eukaryotic cell genetic material which, when expressed, generates a recombinant AEP with cyclization ability, isolating the AEP and incubating the AEP with a linear polypeptide precursor optionally modified to introduce a non-naturally occurring amino acid, the incubation being for a time and under conditions sufficient to generate a cyclic peptide from the polypeptide precursor.
- genetic material encoding the AEP with cyclization ability is co-expressed with genetic material encoding a linear polypeptide precursor in a cell for a time and under conditions sufficient to generate a cyclic peptide in a vacuole or other cellular compartment of the cell.
- This process can also occur in a membranous compartment of a prokaryotic cell such as a periplasmic space.
- the AEP can catalyze a ligation reaction to conjugate two or more peptides wherein at least one peptide comprises a C-terminal AEP recognition amino acid sequence and another peptide comprises an N-terminal AEP recognition amino acid sequence.
- the eukaryotic cell can also be used to generate one or both of the AEP and/or polypeptide precursor for use in the generation of a cyclic peptide in vitro.
- a cyclic peptide can also be produced in the prokaryotic cell.
- the cyclic peptide is produced in the periplasmic space of a prokaryotic cell.
- reference to "peptide” includes a polypeptide or protein. No limitation in size or type of proteinaceous molecule is intended by use of the term "peptide", "polypeptide” or "protein".
- a linear peptide is generated using ligase activity of an AEP.
- a first peptide comprising the C -terminal AEP recognition sequence is co-incubated with a second peptide comprising an N-terminal AEP recognition sequence which may or may not have a tag and an AEP.
- the AEP catalyses a ligation between the first and second peptides to generate a linear peptide conjugate. This may then subsequently be cyclized into a cyclic peptide or used as a linear peptide.
- cyclic peptide includes a “cyclotide”.
- co-incubation means either in vitro in a reaction vessel or in a cell or in a compartment of a cell. Multiple peptides or repeat forms of the same peptides may also be cyclized in vitro or in vivo.
- peptide includes a polypeptide and a protein.
- the AEP is generated in a prokaryotic cell or eukaryotic cell and used in vitro or in vivo to generate a cyclic peptide from a linear polypeptide precursor.
- the AEP and linear polypeptide precursor may also be co-expressed in a prokaryotic cell or eukaryotic cell.
- the linear polypeptide precursor may be produced by synthetic chemistry.
- a recombinant AEP is produced in a prokaryotic or eukaryotic cell, isolated from the cell and used in vitro on any polypeptide precursor to generate a cyclic peptide.
- the genetic material comprises nucleic acid which may be expressed in two respective nucleic acid constructs.
- the recombinant nucleic acid encoding each of the AEP and the polypeptide precursor is expressed in a single nucleic acid construct. Multiple repeats of the same peptide or of different peptides may also be subject to cyclization processing in vivo or in vitro.
- a key aspect is the production of a recombinant form of AEP which is functional having peptide cyclization activity which can either be used in vitro with a precursor polypeptide or a cell expressing an AEP can be used as a recipient for a genetic molecule encoding the precursor polypeptide.
- AEP is a cyclase.
- the set of rules is based inter alia on the presence or absence of residues or gaps in at least 25% of 17 predictive sites. This equates to 5 or more.
- the sites encompass an activity preference loop (APL), active sites and sites proximal thereto and non-active surface residues. Predictive sites are summarized in Table 2.
- a method for determining whether an AEP is likely to have cyclization activity comprising determining the amino acid sequence of the AEP, aligning the sequence with a best fit to the amino acid sequence of OaAEPl b (SEQ ID NO: l) and screening for the presence of 5 or more of residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein Gap means the absence of a residue wherein the presence of 5 or more of the listed residues or absence of residues is indicative of an AEP which is a cyclase.
- a method for determining whether an AEP is unlikely to have cyclization activity comprising determining the amino acid sequence of the AEP, aligning the sequence with a best fit to the amino acid sequence of OaAEPl b (SEQ ID NO: l) and screening for the presence of 13 or more of the residues 139D, 161N, 186G, 192N, 247G, 248T, 253E, 255P, 263T, 293L, residues aligning between residues 299 and 300 of OaAEPl b - N, G, N, Y and S, 314K and 316K wherein the presence of 13 or more of the listed residues is indicative of an AEP which is not a cyclase.
- the AEP may, therefore, be from any source such as but not limited to from the genus Oldenlandia.
- the AEP can be readily tested for cyclase activity.
- One such species is Oldenlandia affinis. Examples include OaAEPl b (SEQ ID NO: l), OaAEPl (SEQ ID NO:2), OaAEP3 (SEQ ID NO:4) or a variant, derivative or hybrid form thereof which retains cyclizing activity.
- the AEP has an amino acid sequence having at least 80% similarity to any one of SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:4 after optimal alignment and wherein the AEP comprises the presence of 5 or more of residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein Gap means the absence of a residue when optimally aligned to SEQ ID NO: l.
- OaAEP2 SEQ ID NO:3
- OaAEP2 SEQ ID NO:3
- the first N-terminal residue in the construct is necessarily methionine.
- an N-terminal methionine precludes cyclization, alternative approaches are utilized. For example:
- a recognition sequence for a protease that cleanly releases the additional residues is added N-terminal to a polypeptide precursor, exposing an appropriate N-terminus for cyclization following cleavage.
- the cyclic peptide has one of a number of activities such as exhibiting pharmaceutical activity and includes an antipathogenic, therapeutic or uterotonic property.
- therapeutic activities include anticancer, protease inhibitory, antiviral or immunomodulatory activity and the treatment of pain.
- the cyclic peptide may also comprise a functional portion fused or embedded in a backbone framework of a cyclotide or other cyclic scaffold (Poth et al. (2013) supra).
- the cyclic peptide may also be generated to be topically applied to a plant or seed of a plant to protect it from pathogen infection or infestation such as against a fungus, bacterium, nematode, mollusc, helminth, virus or protozoan organism. Alternatively, it is topically applied to human or non-human animal surfaces such as a nail, hair or skin.
- the polypeptide precursor may be a natural precursor for the generation of a cyclic peptide or it may not naturally become cyclic but is adapted to generate a cyclic peptide. Such a non-naturally occurring cyclic peptide may, for example, have a longer half life in a composition or when used in vivo or may have greater stability efficacy or utility.
- kits comprising an AEP and a receptacle adapted to receive a polypeptide precursor and means to admix the AEP with the polypeptide precursor. Reagents may also be included to facilitate conversion of the polypeptide precursor into a cyclic peptide.
- the kit contains a eukaryotic or prokaryotic cell comprising genetic material encoding an AEP. Further genetic material encoding a polypeptide precursor to be cyclized is then introduced to that cell.
- An example is a yeast cell such as a Pichia sp.
- the kit enables a useful business model for generating cyclic peptides from any linear polypeptide precursor.
- FIG. 1A is a schematic representation of the Oakl gene product.
- the precursor protein encoded by the Oakl gene (SEQ ID NO:23 encoded by SEQ ID NO:22) is proteolytically processed to produce mature kB l .
- the domains shown in order are: ER signal peptide (ER SP), N-terminal propeptide (NTPP), N-terminal repeat (NTR), cyclotide domain, C-terminal propeptide (CTPP).
- Dashed lines indicate the N- and C-terminal processing sites and a bold asterisk denotes the rOaAEPl cleavage site.
- the C-terminal P3-P1 and ⁇ - ⁇ 3' sites are indicated.
- FIG. 1B is a schematic representation of a synthetic kalata B l precursor carrying the native C-terminal pro-hepta-peptide (GLPSLAA - SEQ ID NO: 10).
- Figure 2 A and B is a Clustal Omega (Sievers et al. (2011) Mol. Syst. Biol 7: 539) alignment of the full-length protein sequences of OaAEPl , Oa ⁇ 3, Oa ⁇ 4 and ⁇ 3 ⁇ 4AEP5.
- FIG. 3 is a graphical representation showing expression of active rOaAEPl b in E. coli.
- FIG. 4 is a representation of the amino acid sequence encoded by the OaAEPl b gene isolated from O. qffinis genomic DNA (SEQ ID NO: l). Predicted ER signal sequence shown in grey; N-terminal propeptide shown in italics; the putative signal peptidase cleavage site is indicated by an open triangle and autocatalytic processing sites are indicated by filled triangles.
- the mature OaAEPl b cyclase domain is underlined. Cys217 and His 175, presumed to be important for catalytic activity, are shown in bold and labeled with an asterisk. The dotted underline indicates possible processing sites for generation of the mature enzyme.
- Figure 5A shows an alignment of the sequence region containing the activity preference loop (APL) for three AEP sequences which act preferentially as proteases (NtAEPlb (SEQ ID NO:55), PxAEP3a (SEQ ID NO:56) and OaAEP2 (SEQ ID NO:3)) and two which act preferentially as cyclases (PxAEP3b (SEQ ID NO:57) and OaAEPl b (SEQ ID NO: l).
- Figure 5B shows an alignment of OaAEPl b (preferentially a cyclase) and OaAEP2 (preferentially a protease) indicating the positions of the 17 cyclase predictive residues (or sites).
- Figure 6 is a graphical representation showing the MALDI MS profile of the enzymatic processing products of a linear kB l precursor (kB l wt ) containing the C-terminal propeptide in the presence of rOaAEPl b .
- Pre linear precursor
- Cyc cyclic product.
- the +6 Da peak corresponds to the reduced form of the cyclic product.
- Figure 7 is a graphical representation showing the kinetics of rOaAEP l b -mediated cyclisation. Varying concentrations of substrate (kB l wt precursor) were incubated with enzyme (19.7 ⁇ g mL 1 total protein) for 5 min. The amount of product formed was inferred by monitoring depletion of the precursor by RP-HPLC. A Michaelis-Menten plot shows the mean of three technical replicates and error bars report the standard error of the mean (SEM). The kinetic parameters derived from this plot are listed (+SEM).
- Figure 8A is a graphical representation of the cyclization by rOaAEPl b (12 ⁇ g mL " 1 total protein) of Bac2A (RLARIVVIRVAR - SEQ ID NO:24), a linear peptide derivative of bactenecin.
- the product was analysed by MALDI MS 22 hours post-addition of rOaAEPl (+ enzyme) or water (- enzyme).
- Bold residues added flanking enzyme recognition sequences.
- Asterisk rOaAEPl cleavage site.
- Observed monoisotopic masses (Da; [M+H] + ) are listed. +22 Da peaks likely represent Na + adducts.
- Cyc cyclic product; Pre, linear precursor.
- Figure 8B is a graphical representation showing the MALDI MS profile of the enzymatic processing products of target peptides with additional AEP recognition residues after 5 h.
- the target peptides shown are (A) the Rl variant GLPVF AEFLPLFS KFGS RMHILKS TRNGL (SEQ ID NO:86), and (B) the Bac2A variant GLPRLARIVVIRVARTRNGLP (SEQ ID NO:87) with bold residues indicating additional AEP residues.
- the enzymes used were (i) rOaAEPl b , (ii) r(3 ⁇ 4AEP3 , (iii) r(3 ⁇ 4AEP4 and (iv) rOaAEP5 and all were at a final concentration of 19.7 ⁇ g mL "1 total protein. A no enzyme control (v) is also shown.
- the expected monoisotopic mass of the cyclized variants are 3074.7 and 2042.3 Da [M+H] + for the Rl variant and the Bac2A variant respectively.
- the observed monoisotopic masses are listed in the figure (Da; [M+H] + ]).
- the +22 Da peak likely represents a sodium adduct.
- Figure 9 is a graphical representation showing the ESI MS profile of the enzymatic processing products of EcAMPl with additional AEP recognition residues (GLPGS GRGSCRS QCMRRHEDEPWRVQEC VS QCRRRRGGGDTRNGLP (SEQ ID NO:60), bold residues indicate additional AEP recognition residues) after 5 h.
- the enzymes used were (i) rOaAEPl b , (ii) r(3 ⁇ 4AEP3, (iii) r(3 ⁇ 4AEP4 and (iv) r(3 ⁇ 4AEP5 and all were at a final concentration of 19.7 ⁇ g mL "1 total protein. A no enzyme control (v) is also shown.
- the expected monoisotopic mass of cyclic EcAMPl is 4892.3 Da. The observed monoisotopic masses are listed in the figure (Da).
- Figure 10 is a graphical representation of the cyclisation of the Rl model peptide with various flanking sequences by bacterially expressed, recombinant AEPs. The proportion of cyclic product is displayed after cyclisation by (A) OaAEPl b (1 h incubation), (B) OaAEP3 (5 h incubation), (C) OaAEP4 (5 hr incubation) or (D) OaAEP5 (1 h incubation).
- FIG 11 is a schematic representation of polypeptide ligation catalyzed by AEPs between a target peptide and a ligation partner peptide.
- the AEP cleavage site is indicated by .
- an AEP cleavage site is incorporated into the target peptide and the ligation partner peptide contains an AEP-compatible N-terminus.
- an AEP cleavage site is incorporated into the ligation partner peptide and the target peptide contains an AEP-compatible N-terminus.
- AEP recognition residues added to the target peptides are shown in bold and the leaving groups are underlined.
- Figure 12 is a graphical representation showing the ESI MS profile of the enzymatic processing products of a target peptide (140 ⁇ ; GLP-NaDl-TRNGLP (SEQ ID NO:79)) and ligation partner peptides (700 ⁇ ) after 6 - 22 h, as indicated.
- the enzymes used were (A) r(3 ⁇ 4AEPl b , (B) r(3 ⁇ 4AEP3, (C) r(3 ⁇ 4AEP4 and (D) r(3 ⁇ 4AEP5 and all were at a final concentration of 19.7 ⁇ g mL "1 total protein.
- the ligation partner was GLPVSGE (SEQ ID NO: 14).
- the ligation partner was PLPVSGE (SEQ ID NO:80).
- no ligation partner was added.
- the labelled NaDl product has the ligation partner peptide added to the C-terminus.
- the expected monoisotopic mass of labelled NaDl is 6641.3 Da when the ligation partner is GLPVSGE and 6681.3 Da when the ligation partner is PLPVSGE.
- the observed monoisotopic masses are listed in the figure (Da).
- Figure 13 is a graphical representation showing the MALDI MS profile of the enzymatic processing products of a target peptide (140 ⁇ ; Rl variant GK VF AEFLPLFS KFGS RMHILKNGL (SEQ ID NO:90)) and a ligation partner peptide (700 ⁇ ; GLK-biotin) after 6 h.
- the enzymes used were (A) r(3 ⁇ 4AEPl b , (B) r(3 ⁇ 4AEP3, (C) r(3 ⁇ 4AEP4 and (D) rOaAEP5 and all were at a final concentration of 19.7 ⁇ g mL "1 total protein.
- the ligation partner peptide was added.
- Figure 14 is a graphical representation showing the MALDI MS profile of the enzymatic processing products of a target peptide (140 ⁇ ; Rl variant GLVF AEFLPLFS KFGS RMHILKGH V (SEQ ID NO:61)) and a ligation partner peptide (700 ⁇ ; biotin-TRNGL) after 6 h.
- the enzymes used were (A) r(3 ⁇ 4AEPl b , (B) rO «AEP3, (C) r(3 ⁇ 4AEP4 and (D) rOaAEP5 and all were at a final concentration of 19.7 ⁇ g mL "1 total protein.
- the ligation partner peptide was added.
- Figure 15 is a graphical representation of the activity of recombinant O. qffinis AEPs ( ⁇ 5 ⁇ g mL "1 total protein) and rhuLEG (1 ⁇ g mL "1 total protein) over time against the fluorogenic substrate Z-AAN-MCA (100 ⁇ ). Activity is tracked at 1 minute intervals at 37°C for 60 minutes using excitation and emission wavelengths of 360 and 460 nm respectively. A single representative experiment is shown. RFU, relative fluorescence units.
- Figure 16 is a graphical representation of r(3 ⁇ 4AEPl b activity against the IQF peptide Abz-STRNGLPS-Y(3N0 2 ) [SEQ ID NO:21] in the presence of protease inhibitors.
- rOaAEPl 4.4 ⁇ g mL "1 total protein
- Enzyme activity against the IQF peptide in the presence of either the Ac- YVAD-CHO or Ac-STRN-CHO inhibitors is reported relative to a no inhibitor control at the 90 minutes time point.
- Figures 17A and 17B are graphical representations of substrate specificity of plant and human AEPs for wt (SEQ ID NO:21) and L31A (SEQ ID NO:25) IQF peptide substrates.
- the assay was conducted at 37°C. The average of two technical replicates are shown and the error bars report the range.
- Figure 18A is a diagrammatic representation of a cyclotide construct for expression in E. coli comprising a cyclotide domain joined via a short linker to ubiquitin- 6xHis. Filled triangle, AEP cleavage site.
- Figure 18B is a diagrammatic representation of an alternative cyclotide construct for expression in E. coli comprising a methionine followed by the kalata B l N-terminal repeat (NTR), cyclotide domain, short linker and ubiquitin-6xHis.
- NTR kalata B l N-terminal repeat
- Figure 19A is a graphical representation showing the MALDI MS profile of the enzymatic processing products of target peptides fused to ubiquitin.
- the target peptides are (A) SFTIl-IlOR-ubiquitin (SEQ ID NO:72) (1 mg mL "1 total protein), (B) kB l-ubiquitin (SEQ ID NO:74) (0.9 mg mL "1 total protein) and (C) Vc 1.1 -ubiquitin (SEQ ID NO:76) (0.24 mg mL "1 total protein).
- Figure 19B is a graphical representation showing enzymatic processing of the kalata B l-ubiquitin fusion protein (SEQ ID NO:74) (260 ⁇ g mL "1 total protein) by different AEPs (19.7 ⁇ g mL "1 total protein) after a 22 h incubation. Approximately 2 ⁇ g of starting material was analysed by SDS-PAGE followed by Western blotting with an anti-6xHis mouse monoclonal antibody.
- Figure 20A is a diagrammatic representation of constructs for Pichia pastoris transformation.
- Construct 1 contains the elements in a single construct and comprises, in sequence, an ER signal sequence, a vacuolar targeting signal (Vac), a cyclotide domain, a short linker and a pro-AEP domain.
- Construct 2 comprises an ER signal sequence, a vacuolar targeting signal, a cyclotide domain and a short linker.
- Construct 3 comprises an ER signal sequence, a vacuolar targeting domain and a pro-AEP domain.
- Constructs 2 and 3 are to be co-transformed. Filled triangles denote AEP cleavage sites; open triangles denote cleavage of the vacuolar targeting signal.
- Figure 20B is a diagrammatic representation of alternative constructs for Pichia pastoris transformation.
- Constructs 4 and 5 are identical to Constructs 1 and 2 respectively ( Figure 20A) except for the addition of a kalata B l N-terminal repeat (NTR) between the vacuolar targeting signal and the cyclotide domain.
- Figure 21 is a graphical representation showing expression of OaAEPl b in Pichia pastoris when kalata B l and AEP were expressed from the same transcriptional unit (SEQ ID NOs: 77 and 78).
- Samples were analysed by SDS-PAGE followed by Western blotting with anti-AEPl b polyclonal rabbit serum. The negative control shows an unrelated protein expressed and extracted under the same conditions.
- T total protein
- L total protein after lysis
- S soluble protein after lysis
- C concentrated soluble protein after lysis
- +ve positive control, rOaAEPl prior to activation.
- Figure 22 is a schematic representation of polypeptide ligation catalyzed by rOaAEPl b between a first peptide (NaDl) having a C-terminal flanking sequence incorporating the rOaAEPl b cleavage site and a 6xHis tag and a second peptide containing an N-terminus compatible with rOaAEPl b .
- the leaving group on the first peptide is underlined.
- cyclic peptide encompasses but is not limited to a "cyclotide”.
- a cyclic peptide is a peptide that is cyclic by virtue of backbone cyclization. It may be naturally cyclic or derived from a non-naturally cyclic linear polypeptide precursor. Hence, the polypeptide precursor from which the peptide is derived may be a natural substrate for cyclization or it may be a naturally linear peptide which is adapted for cyclization.
- peptide includes a polypeptide and a protein.
- cyclic peptide for example, to a "cyclic peptide”, “polypeptide precursor”, “conjugate peptide” and the like is not to exclude a “cyclic polypeptide” or “cyclic protein”, a “precursor peptide” or “precursor protein” or a “conjugate polypeptide” or “conjugate protein”.
- the method comprises the co-incubation either in a receptacle or in a cell of: (i) an AEP with cyclization activity; and (ii) a linear polypeptide precursor of the cyclic peptide.
- the AEP catalyzes the processing of the polypeptide precursor to facilitate excision and circularization of the cyclic peptide. If in a receptacle, the cyclic peptide is purified. If cyclization is catalyzed in a cell, the cyclic peptide is isolated from a vacuole or other compartment within the cell.
- peptide conjugate means two or more peptides ligated together wherein at least one peptide comprises a C -terminal AEP recognition sequence and another peptide comprises an N-terminal AEP recognition sequence.
- the linear polypeptide precursor comprises a C-terminal AEP processing site.
- the C-terminal processing site is an amino acid sequence defined as comprising P3 to PI prior to the actual cleavage site and comprising ⁇ to P3' after the cleavage site towards the C-terminal end.
- P3 to PI and PI to P3 have the amino acid sequence:
- X is an amino acid residue and:
- X 2 is optional or is any amino acid
- X 3 is optional or is any amino acid
- X 4 is N or D
- X 5 is G or S
- X 6 is L or A or I
- X 7 is optional or any amino acid.
- X 2 through X 7 comprise the amino acid sequence:
- the N-terminal end of the linear polypeptide precursor may contain no specific AEP processing site or may contain a processing site defined by any one of PI through P3 wherein PI to P3 is defined by:
- X9 is optional and any amino acid or G, Q, K, V or L;
- X 10 is optional or any amino acid or L, F or I or an hydrophobic amino acid residue
- X 11 is optional and any amino acid.
- X9 through Xn comprise the amino acid sequence: GLX 11
- the AEP processing site comprises N- and C-terminal end sequences comprising the sequence:
- 1 to 2000 includes 1 to 1000 and 1 to 500 such as but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92
- the C-terminal processing site comprises P4 to PI and ⁇ to P4' wherein PI to P4 and ⁇ to P4' comprise X 1 X 2 X 3 X ⁇ 5 X 0 X 7 X 8 wherein X 2 to X 7 are as defined above and X 7 is optional or any amino acid and X 8 is optional or any amino acid.
- the present invention comprises various aspects in relation to the co-incubation of the AEP with cyclization activity and the linear polypeptide precursor which include:
- Aspect (ii) can be modified whereby the linear polypeptide precursor is synthetically produced or isolated from a particular source.
- a linear peptide conjugate can be generated in vitro or in in vivo.
- the AEP and linear polypeptide precursor may be produced in different cells or different cellular compartments of the same cell, isolated then used in vitro.
- the cyclic peptide in a prokaryotic cell, in a non-limiting embodiment, the cyclic peptide is generated by co- expression with an AEP in the periplasmic space.
- the polypeptide precursor may be a natural substrate for cyclization or may normally be a linear peptide that is rendered cyclic. Making a cyclic form of a linear peptide can improve stability, efficacy and utility.
- co-incubation is meant co-incubation in vitro in a receptacle or reaction vessel as well as within a cell.
- the AEP also has ligase activity enabling the generation of peptide conjugates of at least two peptides wherein at least one peptide comprises a C-terminal AEP recognition sequence and at least one other peptide comprising an N-terminal AEP recognition sequence.
- a method for producing a cyclic peptide comprising introducing into the prokaryotic or eukaryotic cell genetic material which, when expressed, generates an AEP with cyclization ability, isolating the AEP and then incubating the AEP with a polypeptide precursor, optionally incorporating a post- translational modification to introduce a non-naturally occurring amino acid residue or cross-linkage bond or other modification for a time and under conditions sufficient to generate a cyclic peptide from the polypeptide precursor; or co-expressing genetic material encoding the AEP with cyclization ability and a linear polypeptide precursor in a prokaryotic or eukaryotic cell for a time and under conditions sufficient to generate a cyclic peptide in a vacuole or other cellular compartment of the cell.
- the AEP can catalyze a ligation reaction to conjugate two or more peptides wherein at least one peptide comprises a C-terminal AEP recognition sequence and another peptide comprises an N-terminal AEP recognition sequence.
- the cell can also be used to generate one or both of the AEP and/or polypeptide precursor for use in the generation of a cyclic peptide in vitro.
- the cyclic peptide is produced by co-expression of an AEP with cyclization ability and a target polypeptide in the periplasmic space of a prokaryotic cell.
- a method of generating a linear peptide conjugate comprising co-incubating two or more peptides wherein at least one peptide comprises a C-terminal AEP recognition sequence and at least one other peptide comprises an N-terminal AEP recognition sequence with an AEP for a time and under conditions sufficient for at least two peptides to ligate together to form a peptide conjugate.
- peptide includes a polypeptide and a protein. No limitation in the size or type of proteinaceous molecule is intended by use of the terms “peptide”, “polypeptide” or “protein”.
- a "vector” refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell.
- the polynucleotide to be delivered comprises a coding sequence of AEP and/or the polypeptide precursor or multiple forms of the same or different peptides.
- the term includes vectors that function primarily for introduction of DNA or RNA into a cell and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.
- a vector in relation to a prokaryotic or eukaryotic cell includes a multi-gene expression vehicle.
- a vehicle consists of a polynucleotide comprising two or more transcription unit segments, each segment encoding an AEP or linear polypeptide precursor, each segment being joined to the next in a linear sequence by a linker segment encoding a linker peptide, the transcription segments all being in the same reading frame operably linked to a single promoter. Multiple polypeptide repeats or multiple different polypeptides may also be generated.
- a vector also includes a viral expression vector which comprises a viral genome with a modified nucleotide sequence which encodes a protein and enable stable expression. Alternatively, multiple vectors are used each encoding either an AEP or linear polypeptide precursor.
- a "transcription unit” is a nucleic acid segment capable of directing transcription of a polynucleotide or fragment thereof.
- a transcription unit comprises a promoter operably linked to the polynucleotide that is to be transcribed, and optionally regulatory sequences located either upstream or downstream of the initiation site or the termination site of the transcribed polynucleotide.
- a single promoter and terminator is used to produce more than one protein from a single transcription unit
- a transcription unit includes a unit encoding either an AEP or a polypeptide precursor, or both.
- a eukaryotic cell includes a yeast, a filamentous fungus and a plant cell.
- a "yeast cell” includes a species of Pichia such as but not limited to Pichia pastoris as well as Saccharomyces or Kluyveromyces.
- Other eukaryotic cells include non-human mammalian cells and insect cells.
- a prokaryotic cell includes an E. coli or some other prokaryotic microorganism suitable for production of recombinant proteins.
- a "host” cell encompasses a prokaryotic cell (e.g. E. coli) or eukaryotic cell (e.g. a yeast cell such as a species of Pichia).
- a prokaryotic cell e.g. E. coli
- eukaryotic cell e.g. a yeast cell such as a species of Pichia.
- nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
- polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from the lineage of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
- the polynucleotide encodes an AEP or linear polypeptide precursor including a linear precursor of a protein to be cyclized or two linear peptides to be ligated or any selectable marker.
- a "gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding an AEP or polypeptide precursor after being transcribed and translated.
- expression refers to the process by which a polynucleotide transcription unit is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as “transcript”) is subsequently translated into an AEP or polypeptide precursor.
- the transcripts and the encoded polypeptides are collectedly referred to as a "gene product”.
- a "linear" sequence is an order of amino acids in the polypeptide in an N- to C-terminal direction in which amino acid residues that neighbour each other in the sequence are contiguous in the primary structure of the polypeptide.
- the "precursor” means it is a substrate for the AEP to generate a cyclic peptide.
- a linear peptide conjugate is generated following ligation of at least two peptides wherein at least one peptide comprises a C-terminal AEP recognition amino acid sequence and at least one peptide comprises an N-terminal AEP recognition amino acid sequence.
- a "pathogen” includes a plant or animal or human pathogen selected from a fungus, insect, bacterium, nematode, helminth, mollusc, virus and a protozoan organism.
- a method for producing a cyclic peptide comprising co-incubating an AEP with peptide cyclizing activity and a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.
- the co-incubation may occur in a receptacle (in vitro) or in a cell such as the vacuole or other cellular compartment of a cell. If the co-incubation is in vitro, then the AEP or the linear polypeptide precursor is produced in a prokaryotic or eukaryotic cell.
- the linear polypeptide precursor may also be produced in a cell and isolated and optionally post- translationally modified or synthetically generated to incorporate a non-naturally occurring amino acid residue or a non-naturally occurring cross-linkage bond or to be isotopically labeled. If co-incubation occurs in a cell, this may occur in a vacuole or other compartment of a eukaryotic cell or in a periplasmic space of a prokaryotic cell.
- AEPs from cyclotide producing plants have been identified that, when expressed with the precursor gene for the cyclotide kalata B l (oakl), and other peptides, are effective at backbone cyclization.
- APL activity preference loop
- ligase competent AEPs the APL either has several residues missing or is replaced by hydrophobic stretch of amino acids ( Figure 5A).
- APL - The absence of residues in the region between 299 - 300 of OaAEPl is predictive of a higher likelihood of cyclase activity.
- APL APL
- set 1 The more of these criteria that it hits, the more likely that it is to be a cyclase.
- Predictive residues for cyclase activity are shown in Table 2. Residue numbering is relative to OaAEPl b ( Figure 4; SEQ ID NO: l). Residue properties that strongly predict cyclase activity are disorder propensity (DISORD), net static charge (CHRG), molecular weight of R group (RMW), and hydropathy index (HPATH).
- DISORD disorder propensity
- CHRG net static charge
- R group R group
- HPATH hydropathy index
- An AEP having at least 25% or 5 or more of the 17 predictive residues set forth in Table 2 is considered likely to act preferentially as a cyclase.
- a requirement for at least 25% of the predictive residues to be present enables 100% of the known cyclases to be correctly identified while excluding known non-cyclases at least 80% of the time including at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 or 94% of the time.
- the rule established herein enables exclusion of non-cyclases 94% of the time.
- One AEP excluded for being a non-cyclase is OaAEP2 (SEQ ID NO:3).
- a method for determining whether an AEP is likely to have cyclization activity comprising determining the amino acid sequence of the AEP, aligning the sequence with a best fit to the amino acid sequence of OaAEPl b (SEQ ID NO: l) and screening for the presence of 5 or more residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein gap means the absence of a residue wherein the presence of 5 or more of the listed residues or absence of residues is indicative of an AEP which is a cyclase.
- the from 5 to 17 residues or gaps screened at the listed sites include 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16 or 17 residues or gaps.
- the AEP is deemed a cyclase.
- At least 25% means 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
- Refernece to "at least 75%” means 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
- a method for determining whether an AEP is unlikely to have cyclization activity comprising determining the amino acid sequence of the AEP, aligning the sequence with a best fit to the amino acid sequence of OaAEPl b (SEQ ID NO: l) and screening for the presence of 13 or more of the residues 139D, 161N, 186G, 192N, 247G, 248T, 253E, 255P, 263T, 293L, residues aligning between residues 299 and 300 of OaAEPlb - N, G, N, Y and S, 314K and 316K wherein the presence of 13 or more of the listed residues is indicative of an AEP which is not a cyclase.
- the present invention extends to any AEP with peptide cyclization activity such as those defined above.
- AEP with peptide cyclization activity
- any other AEP such as, but not limited to, OaAEPlb (SEQ ID NO: l), OaAEPl (SEQ ID NO:2) and OaAEP3 (SEQ IDNO:4) from Oldenlandia qffinis.
- AEPs include an AEP having at least 80% amino acid similarity to SEQ ID NO: l (OaAEPl b ), SEQ ID NO:2 (OaAEPl) or SEQ ID NO:4 (OaAEP3) after optimal alignment and which retains AEP and peptide cyclization activity and when the AEP comrpises the presence of 5 or more of residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein Gap means the absence of a residue when optimally aligned to SEQ ID NO: l.
- the AEP may also have ligase activity to facilitate generation of peptide conjugates.
- OaAEP2 SEQ ID NO:3 is an example of an AEP which is not a cyclase. It is a proviso that statements encompassing cyclase AEPs do not include OaAEP2 (SEQ ID NO:3).
- the first N-terminal residue in a construct is necessarily methionine.
- an N-terminal methionine precludes cyclization, alternative approaches are utilized. For example:
- a recognition sequence for a protease that cleanly releases the additional residues is added N-terminal to the polypeptide precursor, exposing an appropriate N-terminus for cyclization following cleavage.
- Reference to "at least 80%" includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
- similarity includes exact identity between compared sequences at the amino acid level. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, amino acid sequence comparisons are made at the level of identity rather than similarity.
- references to describe sequence relationships between two or more polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
- a “reference sequence” includes from at least 10 amino acid residues (e.g. from 10 to 100 amino acids).
- a “comparison window” refers to a conceptual segment of typically 10 contiguous amino acid residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (BLASTP 2.2.32+, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
- BLASTP 2.2.32+, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA
- Best alignment i.e. resulting in the highest percentage homology over the comparison window
- sequence identity and “sequence similarity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on an amino acid- by-amino acid basis over a window of comparison.
- a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g.
- sequence identity will be understood to mean the "match percentage” calculated by the BLASTP 2.2.32+ computer program using standard defaults. Similar comments apply in relation to sequence similarity.
- a method for producing a cyclic peptide comprising co-incubating an AEP with peptide cyclization activity having an amino acid sequence with at least 80% similarity to a sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and SEQ ID NO:4, after optimal alignment and wherein the presence of 5 or more of residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein Gap means the absence of a residue and a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.
- a method for producing a cyclic peptide in vitro comprising introducing into a prokaryotic cell an expression vector encoding an AEP, enabling expression of the vector to produce a recombinant AEP, isolating the AEP from the cell and co -incubating in a reaction vessel the recombinant AEP with a polypeptide precursor for a time and under conditions sufficient to generate the cyclic peptide.
- Taught herein is a method for producing a cyclic peptide in vitro the method comprising introducing into a prokaryotic or eukaryotic cell an expression vector encoding one or other of an AEP with peptide cyclization activity and a linear polypeptide precursor, enabling expression of the vector to produce a recombinant AEP and recombinant linear polypeptide precursor in the cell or component of the cell or a periplasmic space and isolating a cyclic peptide generated from the polypeptide precursor.
- a method for producing a cyclic peptide in vitro comprising introducing into a prokaryotic or eukaryotic cell an expression vector encoding an AEP with peptide cyclization activity, isolating the AEP and co -incubating in a reaction vessel the AEP with a polypeptide precursor for a time and under conditions sufficient to generate the cyclic peptide.
- the polypeptide precursor may be recombinant or synthetically produced.
- the recombinant polypeptide may be post-translationally modified to introduce, or the synthetic form may incorporate, a non-naturally occurring amino acid.
- a method for producing a cyclic peptide in vivo comprising introduction into a prokaryotic or eukaryotic cell an expression vector encoding an AEP with peptide cyclization activity and a linear polypeptide precursor, enabling expression of the vector to produce the AEP and linear polypeptide precursor to produce a cyclic peptide. In an embodiment, this may occur in a periplasmic space or in a cellular compartment such as a vacuole.
- a method for producing a cyclic peptide in vitro comprising introducing into a prokaryotic or eukaryotic cell an expression vector encoding one or other of an AEP with peptide cyclization activity or a linear polypeptide precursor, enabling expression of the vector to produce a recombinant AEP or recombinant linear polypeptide precursor and isolating the AEP or polypeptide from the cell and co-incubating in a reaction vessel the recombinant AEP with a polypeptide precursor or a post-translationally modified or synthetically modified form thereof for a time and under conditions sufficient to generate the cyclic peptide.
- the AEP comprises an amino acid sequence having at least 80% similarity to any one or more of SEQ ID NOs: l, 2 and/or 4 after optimal alignment.
- reference to "at least 80%" means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
- a linear peptide is generated using the ligase activity of an AEP.
- a first peptide comprising the C-terminal AEP recognition amino acid sequence is co-incubated with a second peptide with an N-terminal AEP recognition amino acid sequence and which may or may not have a tag and an AEP.
- the AEP catalyzes a ligation between the first and second peptides to generate a linear peptide conjugate. This may then subsequently be cyclized into a cyclic peptide or used as a linear peptide. This may occur in vitro or in vivo.
- the polypeptide precursor may be a recombinant molecule generated by expression of nucleic acid encoding same in a cell or a combination of being produced by recombinant means followed by a post-translational modification (e.g. isotopically labeled) or produced by synthetic means. In relation to a post-translation modification or synthetic form, a non- naturally occurring amino acid may be introduced.
- a "cell” includes a prokaryotic (e.g. E. coli) or eukaryotic (e.g. a yeast) cell.
- the nucleic acid encoding the AEP and the polypeptide precursor may be present in two separate nucleic acid constructs or be part of a single construct such as a multi-gene expression vehicle.
- the nucleic acid is operably linked to a promoter which enables expression of the nucleic acid to produce the AEP and/or a linear form of the polypeptide precursor which is then processed into the cyclic peptide either in vitro or in vivo in a vacuole or other cellular compartment.
- cells are maintained which are genetically modified to produce the AEP and these cells are then hosts for any given nucleic acid encoding a polypeptide precursor.
- Taught herein is a method for producing a cyclic peptide in a cell, the method comprising introducing a genetic vector into the cell, the genetic vector comprising polynucleotide segments each encoding either an AEP with peptide cyclization activity or a polypeptide precursor, the polynucleotide segments separated by a polynucleotide linker segment wherein all polynucleotide segments are in the same reading frame operably linked to a single promoter and terminator wherein the eukaryotic cell is grown for a time and under conditions sufficient for a cyclic peptide to be generated which is then isolated from the vacuole or other cellular compartment.
- a method for producing a cyclic peptide in a cell comprising introducing two genetic vectors in the cell, one encoding an AEP with peptide cyclization activity and the other encoding a polypeptide precursor, each genetic molecule comprising a promoter and terminator operably linked to polynucleotides encoding either the AEP or the polypeptide precursor wherein the cell is grown for a time and under conditions sufficient for a cyclic peptide to be generated which is then isolated from the vacuole or other cellular compartment.
- the vector encodes multiple repeats of the same polypeptide to be cyclized or multiple forms of different polypeptides to be cyclized.
- the AEP includes an AEP having at least 80% similarity to one or more of SEQ ID NOs: l, 2 and/or 4 after optimal alignment and wherein the presence of 5 or more of residues or absence of residues at 139K, 161D, 186K, 192D, 247C, 248Y, 253Q, 255A, 263V, 293H, Gap, Gap, Gap, Gap (between residues 299 and 300), 314E and 316G wherein Gap means the absence of a residue.
- OaAEP2 (SEQ ID NO:3) is an example of a non-cyclase AEP.
- a eukaryotic cell includes a yeast cell such as a species of Pichia or a Saccharomyces sp. or Kluyveromyces sp.
- a method for generating a peptide conjugate comprising two or more peptides comprising co-incubating at least two peptides with an AEP wherein at least one peptide comprises a C -terminal AEP recognition amino acid sequence and at least one other peptide comprises an N-terminal AEP recognition amino acid sequence. This may occur in vitro or in vivo.
- Techniques and agents for introducing and selecting for the presence of a vector in cells are well-known. Genetic markers allowing for the selection of the vector cells are well-known, e.g. genes carrying resistance to an antibiotic such as kanamycin, tetracycline or ampicillin. The marker allows for selection of successfully transformed cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene. Eukaryotic cell selection of transformed cells is often accomplished through the inclusion of auxotrophic markers in the vector such as HIS 4 or URA3 which encode enzymes involved in synthesis of essential amino acids or nucleotides.
- auxotrophic markers such as HIS 4 or URA3 which encode enzymes involved in synthesis of essential amino acids or nucleotides.
- vectors are then transformed into a yeast strain that is unable to synthesize specific amino acids or nucleotides that are required for growth, such as histidine for HIS4 and uracil for URA3.
- Cells that have been successfully transformed with the vector are selected by plating on dropout media lacking the specific amino acid or nucleotide as the untransformed cells are not able to synthesize the essential amino acid or nucleotide that is not present in the growth medium whereas cells carrying the vector with the auxotrophic marker survive as they are able to synthesize the missing amino acid or nucleotide.
- Other common auxotrophic markers are LEU2, LYS2, TRP1, HIS3, ARG4, ADE2.
- Techniques for introducing an expression vector comprising a promoter operably linked to a polynucleotide into cell are varied and include transformation, electroporation, microinjection, particle bombardment or other techniques known to the art.
- the choice of vector in which the nucleic acid encoding the AEP or polypeptide precursor is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g. replication, protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant nucleic acid molecules.
- the vector desirably includes a prokaryotic replicon, i.e. a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic cell.
- a prokaryotic replicon i.e. a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic cell.
- replicons are well known in the art.
- the vector could either be maintained extra-chromosomally, in which case the vector sequence would generally comprise a eukaryotic replicon, or could be incorporated into the genomic DNA, in which case the vector would include sequences that would facilitate recombination of the vector into the host chromosome.
- Those vectors that include a prokaryotic replicon also typically include convenient restriction sites for insertion of a recombinant DNA molecule of the present invention.
- Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, CA) and pPL, pK and K223 available from Pharmacia (Piscataway, NJ), and pBLUESCRIPT tm and pBS available from Stratagene (La Jolla, CA).
- a vector of the present invention may also be a Lambda phage vector as known in the art or a Lambda ZAP vector (available from Stratagene La Jolla, CA).
- Another vector includes, for example, pCMU (Nilsson et al. (1989) Cell 58:707-718).
- Other appropriate vectors may also be synthesized, according to known methods; for example, vectors pCMU/Kb and pCMUII used in various applications herein are modifications of pCMUIV (Nilsson et al. (1989) supra).
- the nucleic acid may be DNA or RNA.
- expression of the nucleic acid can be determined using any assay known in the art.
- the presence of a transcribed polynucleotide can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934).
- the polynucleotide encodes the AEP or polypeptide precursor or in the case of a eukaryotic system the polynucleotide may encode both.
- Expression of the nucleic acid can also be determined by examining the protein product.
- a variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunosorbent assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescent assays, and PAGE-SDS.
- mass spectrometry is used for cyclic peptides (Saska et al. (2008) Journal of Chromatography B. 872: 107-114).
- determining the protein level involves (a) providing a biological sample containing polypeptides; and (b) measuring the amount of any immuno specific binding that occurs between an antibody reactive to the AEP or polypeptide precursor, in which the amount of immuno specific binding indicates the level of expressed proteins.
- Antibodies that specifically recognize and bind to AEP or linear polypeptide precursor are required for immunoassays. These may be purchased from commercial vendors or generated and screened using methods well known in the art. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, and Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York.
- the sample of test proteins can be prepared by homogenizing the prokaryotic or eukaryotic transformants and optionally solubilizing the test protein using detergents, such as non-reducing detergents which include triton and digitonin.
- the binding reaction in which the AEP or polypeptide precursor is allowed to interact with the detecting antibodies may be performed in solution, or in cell pellets and/or isolated cells, for example, a solid support that has been immobilized with the test proteins.
- the formation of the complex can be detected by a number of techniques known in the art. For example, the antibodies may be supplied with a label and unreacted antibodies may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. Results obtained using any such assay on a sample from a cell transformant is compared with those from a non-transformed source as a control. Other protein quantitation methods such as BCA and nanodrop methodologies may be employed.
- prokaryotic or eukaryotic host cells of this invention are grown under favorable conditions to effect expression of the polynucleotide.
- prokaryotic cells include E. coli, Salmonella sp, Pseudomonas sp and Bacillus sp.
- eukaryotic cells include yeast such as Pichia spp.(e.g. Pichia pastoris), Saccharomyces spp. or Kluyveromyces spp.
- this invention provides genetically modified cells carrying one or two vectors encoding an AEP and/or a polypeptide precursor.
- the present invention further contemplates a business model for producing cyclic peptides.
- the business model comprises a prokaryotic cell encoding a heterologous AEP with cyclizing activity or a prokaryotic cell for use in introducing and expressing a vector encoding a desired linear polypeptide precursor.
- the polypeptide precursor produced by recombinant or synthetic means and the AEP are co- incubated in a reaction vessel for a time and under conditions sufficient for a cyclic peptide to be generated from the polypeptide precursor.
- a prokaryotic or eukaryotic cell is selected for transformation with a vector encoding an AEP and a polypeptide precursor either in the same or separate constructs or the eukaryotic cell already comprises an AEP-encoding vector and is used as a recipient for a selected vector encoding a polypeptide precursor.
- the cell is then incubated for a time and under conditions sufficient for a cyclic peptide to form which can be isolated from the vacuole of the eukaryotic cell.
- the eukaryotic cell may be used to generate an AEP and/or polypeptide precursor which is used in vitro.
- the business model extends to the generation of linear peptide conjugates.
- the cyclic peptides may have any of a range of useful properties including antipathogen, therapeutic or other pharmaceutically useful properties and/or insecticidal, molluscicidal or nematocidal activity. Examples of therapeutic activities include anticancer, protease inhibitory, antiviral or immunomodulatory activity and the treatment of pain.
- the cyclic peptide may also be a framework to incorporate a functionality.
- a normally linear polypeptide may also be subject to cyclization. This can improve stability, efficacy and utility.
- the polypeptide precursor is a natural substrate for cyclization.
- a non-naturally occurring amino acid may be introduced into the polypeptide precursor.
- These include amino acids with a modified side chain.
- side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
- the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
- the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
- Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
- Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
- Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
- Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
- Examples of incorporating unnatural amino acids and derivatives during polypeptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
- peptides can be conformationally constrained by, for example, incorporation of C and N -methylamino acids, introduction of double bonds between C and C atoms of amino acids.
- the polypeptide precursor may also be isotopically labeled by a cell or during in vitro synthesis.
- a pharmaceutical formulation comprising the cyclic peptide or linear peptide conjugate or a pharmaceutically acceptable salt thereof. Such a formulation has applications in treating human and non-human animal subjects.
- pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the peptides of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
- compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration.
- compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
- Coated condoms, gloves and the like may also be useful.
- compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
- compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
- the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
- Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
- the suspension may also contain stabilizers.
- compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
- the pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
- Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention.
- compositions of the present invention may also include surfactants.
- surfactants used in drug products, formulations and in emulsions is well known in the art.
- the present invention employs various penetration enhancers to effect the efficient delivery of cyclic peptides such as to treat onychomycosis of the nails.
- penetration enhancers also enhance the permeability of keratin.
- Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
- formulations are routinely designed according to their intended use, i.e. route of administration.
- compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
- Dosing is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
- Optimum dosages may vary depending on the relative potency of individual cyclic or linear peptides, and can generally be estimated based on ECso's found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the peptide in bodily fluids or tissues.
- the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the peptide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
- compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
- the cyclic peptide or linear peptide conjugate may also be formulated into an agronomically acceptable composition for topical application to plants or seeds.
- Agronomically acceptable carriers are used to formulate a peptide herein disclosed for the practice of the instant method. Determination of dosages suitable for systemic and surface administration is enabled herein and is within the ordinary level of skill in the art. With proper choice of carrier and suitable manufacturing practice, the compositions such as those formulated as solutions, may be administered to plant surfaces including above- ground parts and/or roots, or as a coating applied to the surfaces of seeds.
- Agronomically useful compositions suitable for use in the system disclosed herein include compositions wherein the active ingredient(s) are contained in an effective amount to achieve the intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.
- compositions for use against plant pathogens may contain suitable agronomically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used in the field, in greenhouses or in the laboratory setting.
- Anti-pathogen formulations include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the cyclic peptides may be prepared as appropriate oily suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Further components can include viscosifiers, gels, wetting agents, ultraviolet protectants, among others.
- Preparations for surface application can be obtained by combining the active peptides with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain powders for direct application or for dissolution prior to spraying on the plants to be protected.
- suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose or starch preparations, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
- disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- the fluorogenic peptide substrate Z-AAN-MCA (where Z is carboxybenzyl and MCA is 7-amido-4- methylcoumarin) was obtained from the Peptide Institute and solubilized in DMSO.
- the inhibitors Ac-YVAD-CHO and Ac-STRN-CHO (where Ac is acetyl and CHO is aldehyde) were synthesized by the Peptide Institute and Mimotopes respectively.
- the linear cyclotide precursor of kalata B l was chemically synthesized and folded as described previously (Simonsen et al. (2004) FEBS Lett 577(3):399-402). This precursor was solubilized in ultrapure water and synthesized with a terminal free acid or amine.
- Figure 1 provides a representation of a linear cyclotide polypeptide precursor.
- Bac2A, EcAMPl and Rl and its derivatives were synthesized with added AEP recognition residues by Genscript or GL Biochem at >85% purity with a terminal free acid or amine and solubilized in ultrapure water, except for one Rl derivative (LL VF AEFLPLFS KFGS RMHILKNGL ; SEQ ID NO:89) which was solubilized in 25% DMSO.
- the ligation partner peptides (GLK-biotin; biotin-TRNGL; GLPVSGE, SEQ ID NO: 14; PLPVSGE, SEQ ID NO: 80) were synthesized by GL Biochem at >85% purity with a terminal free acid or amine and solublilized in ultrapure water.
- Linear target peptides (280 ⁇ , unless otherwise indicated) were incubated with rOaAEPl b , r(3 ⁇ 4AEP3, r(3 ⁇ 4AEP4 or r(3 ⁇ 4AEP5 (total protein concentration as indicated in the description of figures) in activity buffer (50 mM sodium acetate, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.5 ⁇ Tris(2-carboxyethyl)phosphine hydrochloride [TCEP], pH 5). The reaction was allowed to proceed for up to 22 hours at room temperature and was analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), high performance liquid chromatography (HPLC) or nuclear magnetic resonance (NMR) as appropriate.
- MALDI MS matrix-assisted laser desorption/ionization mass spectrometry
- HPLC high performance liquid chromatography
- NMR nuclear magnetic resonance
- Target peptides 140 ⁇ were incubated with a ligation partner peptide (700 ⁇ ) and a recombinant AEP (r(3 ⁇ 4AEPl b , r(3 ⁇ 4AEP3, r(3 ⁇ 4AEP4 or r(3 ⁇ 4AEP5 at 19.7 ⁇ g mL "1 total protein) in activity buffer (50 mM sodium acetate, 50 mM NaCl, 1 mM EDTA, 0.5 ⁇ TCEP, pH 5). The reaction was allowed to proceed for up to 22 hours at room temperature and was analysed by MALDI MS or electrospray ionisation (ESI) mass spectrometry as appropriate.
- activity buffer 50 mM sodium acetate, 50 mM NaCl, 1 mM EDTA, 0.5 ⁇ TCEP, pH 5
- a saturated MALDI matrix solution ( -cyano-4-hyroxycinnamic acid, CHCA, Bruker) prepared in 95% v/v acetonitrile, 0.1% v/v TFA was diluted 1:22 such that the final matrix solution comprised 90% v/v acetonitrile, 0.1% v/v TFA and 1 mM NH 4 H 2 P0 4 .
- Eluted samples were mixed 1:4 with the MALDI matrix, spotted onto a MALDI plate and analyzed by an Ultraflex III TOF/TOF (Bruker) in positive reflector mode. Data were analyzed using the flexAnalysis program (Bruker).
- Polyclonal anti-AEPlb rabbit serum was generated by immunizing a New Zealand White rabbit with a denatured, inactive form of O. affinis AEPlb (residues D47 - P474) that was produced recombinantly in E. coli.
- the rabbit received three doses, four weeks apart of 150 ⁇ g of antigen in 50% (v/v) phosphate-buffered saline (PBS) and Freund's incomplete adjuvant (Sigma). Serum was obtained two weeks after the final dose.
- PBS phosphate-buffered saline
- serum was obtained two weeks after the final dose.
- affinis were present among the contigs (Qin et al. (2010) BMC Genomics 11: 111). Homologues of this AEP sequence were searched using BLAST (Altschul et al. (1990) Mol Biol 215: 403-410) in the contig library using a maximum E-value of le-20, resulting in the identification of 371 putative AEP transcripts. These sequences could then be clustered in 13 groups sharing at least 90% sequence identity using cd-hit (Li et al. (2006) supra). Coding sequences identified were Oa ⁇ 4- 17 (SEQ ID NOs: 39 to 54).
- AEP transcripts from the O. affinis transcriptome assembly were used to design a set of primers.
- a single degenerate forward primer (OaAEPdegen-F, 5 -ATG GTT CGA TAT CYC GCC GG-3 - SEQ ID NO:6) was manually designed to amplify all sequences due to the variability at a single nucleotide position within the 5 region of each full length transcript at the start codon.
- AEP 1-R 5 - TCA TGA ACT AAA TCC TCC ATG GAA AGA GC -3 - SEQ ID NO:7
- ⁇ 3 ⁇ 4AEP2-R 5 - TTA TGC ACT GAA TCC TTT ATG GAG GG -3 - SEQ ID NO:8
- ⁇ 3 ⁇ 4AEP3-R 5 - TTA TGC ACT GAA TCC TCC ATC G -3 - SEQ ID NO:9.
- Target sequences were amplified from the resulting cDNA using Phusion High Fidelity Polymerase (New England BioLabs) and the primers described above under the recommended reaction conditions.
- Gel extracted PCR products were dA-tailed by incubation with Invitrogen Taq Polymerase (Life Technologies) and 0.5 ⁇ ⁇ 10 mM dA in the supplied buffer. The processed products were cloned into pCR8-TOPO (Life Technologies) and transformed into E. coli.
- genomic DNA was extracted from O. qffinis leaf tissue using a DNeasy Plant Mini Kit according to the manufacturer's instructions. PCR amplification from this DNA used primers specifically targeting the OaAEPl nucleotide sequence. Gel extracted product was dA tailed as above, cloned into TOPO (Life Technologies) and transformed into E. coli. DNA from PCR-positive clones was sent for sequencing to the Australian Genome Research Facility. The AEP sequence identified using this method (OaAEPl b ) was subsequently expressed as a recombinant protein.
- AEPs are identified from cyclotide producing plants which, when expressed with the precursor gene for the cyclotide kalata B l (oakl), and other peptides, effect backbone cyclization.
- APL activity preference loop
- APL - The absence of residues in the region between 299 - 300 of OaAEPl is predictive of a higher likelihood of cyclase activity.
- APL APL
- set 1 The more of these criteria that it hits, the more likely that it is to be a cyclase.
- Predictive residues for cyclase activity are shown in Table 2. Residue numbering is relative to OaAEPlb ( Figure 4; SEQ ID NO: l). Residue properties that strongly predict cyclase activity are disorder propensity (DISORD), net static charge (CHRG), molecular weight of R group (RMW), and hydropathy index (HPATH).
- DISORD disorder propensity
- CHRG net static charge
- R group R group
- HPATH hydropathy index
- An AEP having at least 25% (or 5 or more) of the 17 predictive residues set forth in Table 2 is considered likely to act preferentially as a cyclase.
- a requirement for at least 25% of the predictive residues to be present enables 100% of the known cyclases to be correctly identified while excluding known non-cyclases at least 80% including 94%.
- Examples of AEPs predicted to be cyclases using this method include OaAEP4 (88%), OaAEP5 (70%), both sequences derived from transcriptome analysis, which have been tested and shown to be cyclases (e.g. Example 4).
- Other sequences predicted to be cyclases include AEPs from Cicer arietinum (SEQ ID NO:92), Medicago truncatula (SEQ ID NO:93), Hordeum vulgare (SEQ ID NO:94), Gossipium raimondii (SEQ ID NO:95 and Chenopodium quinoa (SEQ ID NO:96) (Example 10).
- DNA encoding full-length O. affinis AEPs without the putative signalling domain (OaAEPlb residues A 2 7-P 4 78, Oa ⁇ 3 residues R 28 -A 4 9i, Oa ⁇ 4 residues A 28 -A 4 9i or (3 ⁇ 4AEP5 residues E 27 -L 48 5) was inserted into the pHUE vector (Catanzariti et al.
- Transformed cells were grown at 30°C in superbroth (3.5% tryptone [w/v], 2% yeast extract [w/v], 1% glucose [w/v], 90 mM NaCl, 5 mM NaOH) to mid-log phase; the temperature was then reduced to 16°C and expression was induced with isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG; 0.4 mM; Bio Vectra) for approximately 20 hours. Cells were harvested by centrifugation and resuspended in non-denaturing lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% triton X 100, lmM EDTA, pH 7).
- non-denaturing lysis buffer 50 mM Tris-HCl, 150 mM NaCl, 0.1% triton X 100, lmM EDTA, pH 7).
- Lysis was promoted by a total of five freeze/thaw cycles and the addition of lysozyme (hen egg white; Roche; 0.4 mg mL 1 ). DNase (bovine pancreas; Roche; 40 ⁇ g mL -1 ) and MgCl 2 (0.4 M) were also added. Cellular debris was removed by centrifugation and the lysate was stored at -80°C until required.
- lysozyme hen egg white; Roche; 0.4 mg mL 1
- DNase bovine pancreas; Roche; 40 ⁇ g mL -1
- MgCl 2 0.4 M
- Lysate containing expressed recombinant AEPs was filtered through a 0.1 ⁇ glass fibre filter (GE Healthcare) before being diluted 1:8 in buffer A (20 mM bis-Tris, 0.2 M NaCl, pH 7) and loaded onto two 5 mL HiTrap Q Sepharose high performance columns connected in series (GE Healthcare; 1.6 - 3.1 mL undiluted lysate mL "1 resin).
- Bound proteins were eluted with a continuous salt gradient (0-30% buffer B [20 mM bis-Tris, 2 M NaCl, pH 7]; 15 column volumes [cv]) and AEP-positive fractions identified by Western blotting (anti-AEPlb rabbit serum [1:2000]; peroxidase-conjugated anti-rabbit IgG [GE Healthcare; 1:5000]).
- AEPs are usually produced as zymogens that are self -processed at low pH to their mature, active form (Hiraiwa et al. (1997) Plant J 12(4 ):S19-S29; Hiraiwa et al.
- Protein precipitation at pH 4.5 allowed removal of the bulk of the contaminating proteins by centrifugation. The remaining protein was filtered (0.22 ⁇ ; Millipore), diluted 1:8 in buffer A2 (50 mM acetate, pH 4) then captured on a 1 mL HiTrap SP Sepharose high performance column (GE Healthcare). Bound proteins were eluted with a salt gradient (0-100% buffer B2 [50 mM acetate, 1 M NaCl, pH 4]; 10 cv) and fractions with activity against an IQF peptide (Abz-STRNGLPS-Y(3N0 2 )) were pooled and used in subsequent activity assays.
- Figure 3B shows that after activation of rOaAEPl b , a dominant band of -32 kDa was evident by reducing SDS-PAGE and Instant blue staining (Expedeon) and this was confirmed to be rOaAEPl by Western blotting.
- Experimentally determined self-processing sites of rOaAEPl b are indicated in Figure 4. The total concentration of protein in each preparation was estimated by BCA assay according to the manufacturer's instructions.
- a volume of 700 1 was loaded onto a reversed-phase C18 analytical column (Agilent Eclipse C18, 5 m, 4.6 x 150 mm) and peptides were separated by HPLC (19 min linear gradient of 12-60% acetonitrile, 0.1% TFA at 1 ml min -1 ). The identity of eluted peaks was confirmed using MALDI MS. The area under the curve corresponding to the precursor peptide was quantitated by comparison to a standard curve and initial velocities were calculated by converting this to moles product formed. Kinetic parameters were estimated using the Michaelis-Menten equation and the curve-fitting program GraphPad Prism (GraphPad Software, San Diego).
- Rl was used as a model peptide.
- the recognition residues added to this model peptide were sequentially trimmed to determine the minimal requirements for AEP-mediated cyclization.
- the N- and C-terminal recognition residues were also substituted with alternate amino acids to determine if particular classes of residues were preferred for cyclization by these recombinant AEPs.
- the ability of activated AEPs (rOaAEPl b , r(3 ⁇ 4AEP3, r(3 ⁇ 4AEP4 and r(3 ⁇ 4AEP5) to cyclize the Rl peptide with varied flanking sequences was then tested in the cyclization assay described in the Materials and Methods ( Figure 10; Table 3).
- Target peptides were incubated with ligation partner peptides as well as active enzyme (Figure 11). The appearance of new linear peptides were tracked.
- Ligation of labeled peptides to a target polypeptide provides a generic, targeted protein labeling strategy for a variety of moieties (e.g. fluorescent labels, biotin, affinity tags, epitope tags, solubility tags) that is limited only by the ability of synthetic peptide chemistry or other methods to produce the appropriate ligation partner. This approach can also enable ligation of multiple domains that could be challenging to produce as a single recombinant protein.
- AEP recognition residues were added to the N- and C-termini of the plant defensin NaDl (Lay et al. (2003) Plant Physiol 131: 1283- 1293) to produce a modified defensin (GLP-NaDl-TRNGLP; SEQ ID NO:79). This was recombinantly expressed in Pichia pastoris and purified using a similar method to that described in Lay et al. (2012) Biol Chem 287: 19961-19972.
- a modified Rl peptide, (GL VF AEFLPLFS KFGS RMHILKGHV ; SEQ ID NO:61) that was not itself an AEP substrate since it does not contain the required Asx residue was used as a target peptide for N-terminal labelling.
- the Rl derivative was incubated with a biotinylated ligation partner peptide (biotin-TRNGL; SEQ ID NO: 104) and recombinant AEPs.
- MALDI-MS showed that AEP-mediated processing created a new linear peptide that incorporated an N-terminal biotin ( Figure 14).
- AEP activity has traditionally been tracked by monitoring cleavage of the fluorescent substrate Z-AAN-MCA (where Z is carboxybenzyl; MCA is 7-amido-4- methylcoumarin) [Saska et al. (2007) supra; Rotari et al. (2001) Biol. Chem. 382:953- 959].
- Cleavage C-terminal to the Asn liberates the fluorophore which then fluoresces to report substrate cleavage.
- butelase-1 Nguyen et al.
- IQF peptides that incorporate the P1-P4 as well as the ⁇ - ⁇ 4' residues are, however, effectively targeted by recombinant O. qffinis AEPs. These peptides contain a fluorescence donor/quencher pair, with fluorescence observed upon the spatial separation of this pair following enzymatic cleavage. Activity against such IQF reporter peptides without corresponding activity against the generic substrate (Z-AAN-MCA) may allow rapid identification of members of the AEP family likely to have cyclizing ability.
- rOaAEPl displayed a preference for a bulky hydrophobic residue such as Leu at the P2' position that was not shared by human legumain (rhuLEG), an AEP that preferentially functions as a hydrolase ( Figures 17A and 17B).
- P2' specificity could also be used to predict cyclization ability and or to select AEPs with different sequence requirements in the substrate to be cyclized EXAMPLE 7
- DNA encoding a target peptide for cyclisation, a short linker (Glu-Phe-Glu-Leu or Gly-Gly-Gly-Gly-Ser-Glu-Phe-Glu-Leu) and a C-terminal ubiquitin-6xHis was inserted into either the pHUE vector (Catanzariti et al. (2004) supra) ⁇ Xbal/BamHI) or the pET23a(+) vector (Invitrogen; Ndel/Xhol) to give a target peptide-linker-ubiquitin-6xHis fusion protein construct ( Figure 18 A).
- the linker coding region contains restriction sites for easy substitution of the target peptide domain with other target sequences.
- the DNA sequence inserted included nucleotides prior to the initiating Met codon to ensure the original vector sequence was reconstituted. If not naturally present in the target peptide, appropriate N-and C-terminal AEP recognition sequences were introduced. Since the first residue of all target peptides was necessarily Met, the N- terminal recognition sequence added was Met-Leu. The C-terminal recognition sequence was Asn-Gly-Leu-Pro.
- the target peptide is preceeded by an initiating Met followed by the kalata B l N-terminal repeat (NTR) ( Figure 18 B) or other cleavable domain.
- the target peptides produced as fusion proteins with ubiquitin were the cyclotide kB l (SEQ ID NO:74), the modified sunflower trypsin inhibitor SFTI-1 I10R (Quimbar et al. (2013) Biol Chem 288( 9): 13885-13896) (SEQ ID NO:72) and the conotoxin Vcl.l (Clark et al. (2010) supra) (SEQ ID NO:76).
- the constructs were introduced into T7 Shuffle E. coli cells (New England BioLabs) and grown at 30°C in 2YT (16% [w/v] tryptone, 10% [w/v] yeast, 5% [w/v] sodium chloride) to mid-log phase.
- the lysate was then filtered through a 0.1 ⁇ glass fibre filter (GE Healthcare) and passed over a Ni-NTA resin (QIAgen) to capture 6xHis tag protein.
- Bound protein was eluted with elution buffer (50mM sodium phosphate pH 8.0, 300 mM NaCl, 250 mM imidazole) and the total protein concentration was estimated by BCA assay according to the manufacturer's instructions. Fusion proteins were then used in enzyme assays.
- the eluted protein is first buffer exchanged into water or appropriate buffer before AEP processing is assayed.
- the eluted protein is first further purified by diluting 1: 10 in 20 mM Tris-HCl, pH8 and passing over as second resin (Q sepharose high performance anion exchanger; GE Healthcare).
- Bound protein is recovered by a continuous salt gradient (0-100% 20 mM Tris-HCl, 1M NaCl pH 8), optionally buffer exchanged into ultrapure water or appropriate buffer and concentrated.
- AEPs r(3 ⁇ 4AEPl b , r(3 ⁇ 4AEP3, r(3 ⁇ 4AEP4, r(3 ⁇ 4AEP5) to release the ubiquitin tag and cyclise target peptides was investigated using the cyclisation assay described in the Materials and Methods followed by MALDI MS ( Figure 19 A, Table 4). Estimated substrate and enzyme concentrations are as indicated in the description of the figures. When required, 3.3% v/v glacial acetic acid was also added to the reaction mix to ensure the assay was carried out at acidic pH.
- the mixture is then diluted 1:5 in non-denaturing lysis buffer and again passed over a Ni-NTA resin (QIAgen).
- QIAgen Ni-NTA resin
- the processed, cyclic, product no longer contains a 6xHis tag and is therefore present in the unbound fraction.
- This product is then dialysed into ultrapure water, concentrated and analysed by MALDI MS, HPLC or NMR to confirm its cyclic structure.
- an NTR is included between the VTR and the cyclotide domain ( Figure 20B, construct 4) or residues Meti-Lysios of the P. pastoris carboxypeptidase Y sequence are included in the construct described above.
- a linker region (Ala-Ala-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Ser - SEQ ID NO: 18) was included between kalata B l and OaAEPl b to reduce steric hindrance between the cyclotide and AEP domains at the protein level and introduce restriction sites for easy substitution of the cyclotide domain with DNA sequences encoding other target peptides.
- Linkers could incorporate the MGEV linker (Glu-Glu-Lys-Lys-Asn - SEQ ID NO: 17) or an extended sequence (e.g.Ala-Ala-Ala-[Gly-Gly-Gly-Gly-Gly-Ser] 2"5 ).
- the foreign DNA was then introduced into GS 115 P. pastoris cells.
- the vector encoding kalata B l and OaAEPl was then linearized by restriction digestion with Sail and introduced into GS 115 cells where it was integrated into the genome at the his4 locus.
- Kalata B l and OaAEPl b were also expressed from separate transcriptional units ( Figure 20 A, constructs 2 and 3).
- DNA encoding an ER signal sequence and a vacuolar targeting sequence (P. pastoris carboxypeptidase Y, residues Meti-Valio 7 ) and kalata B l (including a short C-terminal tail [Gly-Leu-Pro]) ( Figure 20 A, construct 2) was inserted into pPICZa (such that the alpha mating factor secretion signal was cloned out and replaced with the ER signal sequence and vacuolar targeting sequence).
- the vector was then linearized with Sacl and introduced into GS115 cells where it was integrated into the genome at the 5' AOX1 locus.
- the cyclotide domain is preceded by an NTR inserted C-terminally to the vacuolar targeting sequence ( Figure 20 B, construct 5).
- DNA encoding an ER signal sequence and a vacuolar targeting sequence (P. pastoris carboxypeptidase Y, residues Meti-Valio?) and OaAEPlb ( Figure 20 A, construct 3) was inserted into pPIC9 (such that the alpha mating factor secretion signal was cloned out and replaced with the ER signal sequence and the vacuolar targeting sequence).
- the vector was then linearized by restriction digestion with Sail and introduced into GS 115 cells already harboring the kalata B l construct.
- the OaAEPl b construct was integrated into the genome at the his4 locus.
- GS 115 cells harboring the appropriate construct/s were grown in 5 mL buffered minimal glycerol medium (BMG; 10 mM potassium phosphate, pH 6, 0.34% w/v yeast nitrogen base, 4 x 10 "5 % w/v biotin, 1% v/v glycerol) at 30°C, with shaking, for 48 hours.
- BMG buffered minimal glycerol medium
- This starter culture was then used to inoculate 40 mL of BMG and grown at 30°C, with shaking, overnight.
- Cells were harvested by centrifugation and resuspended in 200 mL buffered methanol medium (BMM; 10 mM potassium phosphate, pH 6, 0.34% w/v yeast nitrogen base, 4 x 10 "5 % w/v biotin, 1% v/v methanol) to induce recombinant protein expression.
- BMM buffered methanol medium
- the culture was incubated at 30°C, with shaking, for 72 hours and methanol was added to 0.5% every 24 hours. After 72 hours, cells were harvested by centrifugation and resuspended in breaking buffer (30 mM HEPES, pH 7.4, 500 mM NaCl) (Visweswaraiah et al. (2011) J. Biol. Chem.
- the vacuolar targeting signal is added to facilitate trafficking of the expressed proteins to the vacuole of Pichia pastoris and the in vivo cyclization of target peptides.
- the cyclic target peptides are then directly purified from the cells. This could be aided by isolation of the vacuolar fraction. This is carried out as previously described (Cabrera and Ungermann (2008) Methods Enzymol 451: 177-196). Volumes relate to a 1 L culture at OD 6 oo and are scaled accordingly. Thawed cells are resuspended in 33.3 mL 0.1M Tris- HCl, pH 9.4, 10 mM dithiothreitol (DTT) and incubated at 30°C for 10 minutes.
- DTT dithiothreitol
- spheroplasting buffer (0.18 x YPD [0.18% w/v yeast extract, 0.36% w/v bactopeptone, 0.36% w/v dextrose, pH 5.5], 240 mM sorbitol, 50 mM potassium phosphate pH 7.5).
- a further 3.3 mL of spheroplasting buffer combined with lyticase (Sigma, as per manufacturer's instructions) is then added and cells are incubated at 30°C, 20 minutes.
- Cells are harvested by centrifugation and resuspended in 1.67 mL 15% Ficoll (w/v in PS buffer [10 mM PIPES/KOH, pH 6.8, 200 mM sorbitol]).
- Dextran solution (10 mg mL "1 DEAE-dextran, 10 mM PIPES/KOH, pH 6.8, 200 mM sorbitol) is added to 0.4 mg mL "1 and cells are incubated on ice (5 minutes), 30°C (1.5 minutes), and ice again (5 minutes).
- Cell lysates are transferred to centrifuge tubes and sequentially layered with 3 mL of 8% w/v Ficoll (in PS buffer), 4% w/v Ficoll (in PS buffer) and PS buffer. The lysate is centrifuged at 110,000xg at 4°C for 90 minutes and vacuoles are collected from the 0-4% w/v Ficoll interface.
- Isolated vacuoles are osmotically lysed (Wiederhold et al. (2009) Mol Cell Proteomics 8:380-392) by addition of a four-fold volume of 20 mM Tris-HCl, pH 8, 10 mM MgCl 2 , 50 mM KC1 (30 minutes, 4°C with agitation).
- the lysed vacuoles are filtered through a 0.22 ⁇ filter, further diluted 1:4 with 20 mM Tris-HCl, pH 8 and bound to a Q sepharose high performance anion exchange resin (GE Healthcare).
- Bound kalata B l is recovered by a continuous salt gradient (0-100% 20 mM Tris-HCl, 1M NaCl pH 8), buffer exchanged into ultrapure water and concentrated.
- the sample is loaded onto an Agilent Zorbax C18 reversed-phase column (4.6 x 250 mm, 300 A) and separated using a linear gradient of 5-55% buffer B (90% acetonitrile, 10% v/v H 2 0, 0.05% v/v TFA) in buffer A (0.05% v/v TFA/H 2 0) over 60 minutes.
- Fractions containing kalata B l are lyophilized, resuspended in ultrapure water and analyzed by MALDI MS, HPLC or NMR to confirm its cyclic structure.
- cyclized proteins are generally more stable than linear proteins a crude extract could also be heated to 70°C for 1 hour after cell disruption and centrifuged at 4000g for 20 minutes to denature and remove the majority of non-cyclized cellular protein. Cyclized protein will then be purified from the cleared extract as described below for the vacuolar extract.
- the appearance of the ligation product (NaDl- STRNGLPVSGEK-FITC - SEQ ID NO: 16 or NaD 1 -STRNGLPVSGE- SEQ ID NO: 15) is tracked by MALDI MS.
- the mixture is diluted 1:5 in non-denaturing lysis buffer (without triton X 100) and passed over a Ni- NTA resin (QIAgen).
- the ligated product does not contain a 6xHis tag and is therefore present in the unbound fraction.
- AEPs from Cicer arietinum (SEQ ID NO: 92), Medicago truncatula (SEQ ID NO: 93), Hordeum vulgare (SEQ ID NO: 94), Gossypium raimondii (SEQ ID NO: 95) and Chenopodium quinoa (SEQ ID NO: 96) are recombinantly expressed in E. coli.
- DNA encoding these full-length AEPs without the putative signalling domain (CaAEP residues Q56-P 4 60, iAEP residues Es 4 -N 4 97, HvAEP residues G60-Y508, GrAEP residues Q31-H500, CqAEP residues R33-V599) is inserted into the pHUE vector (Catanzariti et al. (2004) supra) to give a 6xHis-ubiquitin-AEP fusion protein construct. Residue numbering is as determined by a multiple alignment of the five sequences generated using Clustal Omega (Sievers et al. (2011) supra). DNA is then introduced into T7 Shuffle E. coli cells (New England BioLabs).
- Transformed cells are grown at 30°C in superbroth (3.5% tryptone [w/v], 2% yeast extract [w/v], 1% glucose [w/v], 90 mM NaCl, 5 mM NaOH) to mid-log phase; the temperature is then reduced to 16°C and expression is induced with isopropyl ⁇ - D-l-thiogalactopyranoside (IPTG; 0.4 mM; Bio Vectra) for approximately 20 hours. Cells are harvested by centrifugation and resuspended in non-denaturing lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% triton X 100, ImM EDTA, pH 7).
- non-denaturing lysis buffer 50 mM Tris-HCl, 150 mM NaCl, 0.1% triton X 100, ImM EDTA, pH 7).
- Lysis is promoted by a total of five freeze/thaw cycles and the addition of lysozyme (hen egg white; Roche; 0.4 mg mL "1 ).
- DNase bovine pancreas; Roche; 40 ⁇ g mL "1 ) and MgCl 2 (0.4 M) are also added. Cellular debris is removed by centrifugation and the lysate is stored at -80°C until required.
- Lysate containing expressed recombinant AEPs is filtered through a 0.1 ⁇ glass fibre filter (GE Healthcare) before being diluted 1:8 in buffer A (20 mM bis-Tris, 0.2 M NaCl, pH 7) and loaded onto two 5 mL HiTrap Q Sepharose high performance columns connected in series (GE Healthcare; 1.6 - 3.1 mL undiluted lysate mL "1 resin).
- Bound proteins are eluted with a continuous salt gradient (0-30% buffer B [20 mM bis-Tris, 2 M NaCl, pH 7]; 15 column volumes [cv]) and AEP-positive fractions are identified by Western blotting (anti-AEPlb rabbit serum [1:2000]; peroxidase-conjugated anti-rabbit IgG [GE Healthcare; 1:5000]).
- AEPs are usually produced as zymogens that are self -processed at low pH to their mature, active form (Hiraiwa et al. (1997) supra; Hiraiwa et al. (1999) supra; Kuroyanagi et al. (2002) supra).
- EDTA 1 mM
- TCEP Sigma- Aldrich; 0.5 mM
- the pH is adjusted to 4.5 with glacial acetic acid and the protein pool is incubated for 5 hours at 37°C.
- Protein precipitation at pH 4.5 allows removal of the bulk of the contaminating proteins by centrifugation.
- the remaining protein is filtered (0.22 ⁇ ; Millipore), diluted 1:8 in buffer A2 (50 mM acetate, pH 4) then captured on a 1 mL HiTrap SP Sepharose high performance column (GE Healthcare).
- Bound proteins are eluted with a salt gradient (0-100% buffer B2 [50 mM acetate, 1 M NaCl, pH 4]; 10 cv) and fractions with activity against an IQF peptide (Abz-STRNGLPS-Y(3N0 2 ) or other target sequence or fluorescent peptide as appropriate) are pooled and used in subsequent activity assays.
- the total concentration of protein in each preparation is estimated by BCA assay according to the manufacturer's instructions.
- Enzymes are used in cyclization and ligation assays as described in the Materials and Methods.
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EP3515931A4 (en) * | 2016-09-23 | 2020-04-22 | Nanyang Technological University | Methods for enzymatic peptide ligation |
US11795488B2 (en) | 2016-09-23 | 2023-10-24 | Nanyang Technological University | Methods for enzymatic peptide ligation |
WO2020226572A1 (en) * | 2019-05-07 | 2020-11-12 | Nanyang Technological University | Asx-specific protein ligases and uses thereof |
US20220213461A1 (en) * | 2019-05-07 | 2022-07-07 | Nanyang Technological University | Asx-specific protein ligases and uses thereof |
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EP3353315A1 (en) | 2018-08-01 |
EP3353315A4 (en) | 2019-10-16 |
JP2019500879A (en) | 2019-01-17 |
US20190225652A1 (en) | 2019-07-25 |
AU2016327453A1 (en) | 2018-04-26 |
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