WO1998050563A1 - Methodes de production d'un peptide amide par utilisation d'une proteine de fusion - Google Patents

Methodes de production d'un peptide amide par utilisation d'une proteine de fusion Download PDF

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WO1998050563A1
WO1998050563A1 PCT/GB1998/001281 GB9801281W WO9850563A1 WO 1998050563 A1 WO1998050563 A1 WO 1998050563A1 GB 9801281 W GB9801281 W GB 9801281W WO 9850563 A1 WO9850563 A1 WO 9850563A1
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Prior art keywords
fusion protein
peptide
expressed
sequence
transgenic
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PCT/GB1998/001281
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English (en)
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Ian Robert Cottingham
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Ppl Therapeutics (Scotland) Ltd.
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Priority to CA002287204A priority Critical patent/CA2287204A1/fr
Priority to EP98919369A priority patent/EP0979291A1/fr
Priority to JP54783598A priority patent/JP2001525664A/ja
Priority to KR19997010114A priority patent/KR20010012165A/ko
Priority to NZ500507A priority patent/NZ500507A/en
Priority to AU72244/98A priority patent/AU7224498A/en
Publication of WO1998050563A1 publication Critical patent/WO1998050563A1/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06B—TREATING TEXTILE MATERIALS USING LIQUIDS, GASES OR VAPOURS
    • D06B1/00—Applying liquids, gases or vapours onto textile materials to effect treatment, e.g. washing, dyeing, bleaching, sizing or impregnating
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57527—Calcitonin gene related peptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06C—FINISHING, DRESSING, TENTERING OR STRETCHING TEXTILE FABRICS
    • D06C2700/00—Finishing or decoration of textile materials, except for bleaching, dyeing, printing, mercerising, washing or fulling
    • D06C2700/13—Steaming or decatising of fabrics or yarns
    • D06C2700/135—Moistening of fabrics or yarns as a complementary treatment

Definitions

  • the present invention is directed to the production of peptides, especially but not exclusively with carboxy-terminal modifications such as amidation, by recombinant means. 5
  • Peptide is a term loosely applied to a chain of amino acids, arbitrarily applied to sequences of three to over one hundred components, but possibly more, joined via their amino- and carboxy teraiini.
  • Naturally occurring peptides which function as hormones, messengers, growth factors, antimicrobials, 0 surfactants etc and a wide variety of medicinal and other applications can be envisaged.
  • a wide panel of protein cleavage technologies can be envisaged. These range from chemical cleavage at specific amino acids to enzymatic cleavage using sequence-specific enzymes. Examples of chemical cleavage include cyanogen bromide cleavage after methionine residues and hydroxylamine cleavage between the amino acid pair asparagine - glycine. Examples of enzymes suitable for cutting at specific protein sequences include enterokinase, which cuts after the sequence (aspartic acid) 4 -lysine, and thrombin, which cuts after the basic amino acids lysine or arginine.
  • a common problem with both of these cleavage strategies is that sequence constraints operate on both the presence of internal sites within the peptide and the necessity to generate authentic ammo-termini. For example, cyanogen bromide is only useful when there are no internal methionines in the peptide and thrombin can cut at a number of different sites after basic amino acids. Enzymatic cleavage has additional problems in terms of process economics. The enzyme must come from an acceptable and validated source (a common source of enterokinase is calf gut endothelium) and be available in economically acceptable quantity.
  • Carboxy-terminal amidation is a common post-translational modification found on many biologically active peptides of potential commercial interest. Examples include calcitonin, magainin and etc. . In many instances, for example, calcitonin, the natural amidated peptide is nearly two thousand times as active as the non-amidated version.
  • This invention describes a method for the production of peptides as ammo-terminal extensions of fusion proteins in recombinant systems.
  • We provide novel methods whereby cleavage of the peptide from the fusion protein and modifications of the peptide such as carboxy-amidation can occur as a series of linked reactions in a single process.
  • Such an approach benefits from the low cost and fidelity of synthesis in a biological expression system without the disadvantages posed by the necessity of a separate cleavage step.
  • the present invention provides a method for the production of a peptide which comprises the step of expressing the peptide as part of a fusion protein followed by release of the peptide from the fusion protein by an acyl- acceptor such as a sulphur containing reductant.
  • an acyl- acceptor such as a sulphur containing reductant.
  • at least part of the fusion protein is a molecule capable of catalysing transfer of the peptide, as an acyl moiety, to a suitable acceptor such as a proximal sulphur atom to form the thio-ester.
  • the peptide is chemically modified, eg amidated at its carboxy terminus after release from the fusion protein.
  • the amidation step is carried out in the presence of a source of ammonium ions at a suitable pH and the amidation step occurs simultaneously with release of the peptide.
  • amidated peptides which could be prepared using these methods include Salmon Calcitonin, Human Calcitonin, Lutenising hormone releasing hormone, Oxytocin, Gastrin neuropeptide Y, Vasopressin, Corticotrophin releasing hormone, Growth hormone releasing hormone, Human Calcitonin gene related peptide, Gastrin, D- tyr-trp-gly, phe-gly-phe-gly, gly-phe-gly, Melanocyte stimulation hormone precursor, Sectetin, Thyrotrophin releasing hormone, Amylin, Substance P, Pancreatic polypeptide, Cholecystokinin, Gastrin secretion factor, phe-his-ile, phe- tyr-tyr, Savagin, Mastoparin M, Caerulein and FMRF amide.
  • the methods of the present invention can for example utilise a commercially available expression vector designed for making proteins as fusion proteins.
  • This vector incorporates a modified self-splicing protein, an intein, making it possible to liberate the protein from its fusion partner by a simple chemical reaction.
  • the invention utilises modified chemical conditions/steps to result in cleavage of the fusion protein thereby liberating a desired peptide, which can be modified e.g. by ambition at the carboxy-terminus.
  • Inteins are proteins which are expressed with flanking protein sequences at both amino- and carboy-te ⁇ riini.
  • the amino- and carboxy-terminal sequences have been named exteins in keeping with the DNA nomenclature of exons and introns.
  • a seemingly typical member of the emerging family of inteins is the VMA1 gene product from yeast. This is approximately 50kDa in molecular mass and contains essential amino acids at the amino terminal (Cysteine) and at the carboxy-terminal (histamine and asparagine).
  • the carboxy-terminal extein must start with a cysteine.
  • ammo-terminal peptide bond is broken and the extein transferred to the sulphur atom of the adjacent cysteine to form a thio-ester. This bond is then exchanged with the cysteine at the start of the carboxy-terminal extein and then, with participation of the adjacent asparagine, exchanged with the peptide bond at this end of the intein.
  • the overall effect of these conceited reactions is that the two exteins are seamlessly joined and the intein is released.
  • Calcitonin is an example of a medically and commercially important peptide suitable for manufacture using the methods described in this invention. It contains thirty-two amino acids and is amidated at the carboxy-terminus. The functional activity and amino acid sequence is highly conserved between species. Thus salmon Calcitonin , which was originally obtained mostly from natural sources but is now made by direct synthesis, is in widespread clinical use. In the past, therapies have focused on Paget's disease and hypocalcaemic shock. However, recently there has been a demand for larger amounts of material to treat osteoporosis in post-menopausal women. This application requires substantive quantities of material which makes the cost of production an increasingly important factor.
  • oligonucleotides which encode the Calcitonin sequence flanked by restriction sites designed for insertion at the appropriate site 5' to the modified intein. These sites must be chosen so that the coding sequence of the peptide is in the same coding frame as the rest of the expressed protein. Suitable oligonucleotides can be made by any number of methods, known to those skilled in the art, including most obviously direct synthesis and polymerase chain reaction amplification from a natural sequence using primers designed to contain convenient restriction sites. This DNA construct is then transformed into a suitable expression system and the resulting fusion protein harvested.
  • the fusion protein also comprises a label, which allows for identification and/or purification of the fusion protein, and thus the peptide, by affinity or other chromatographic methods.
  • a suitable label include a specific chitin-binding domain, or part thereof, a repeat of acidic or basic amino acids, a poly-histidine sequence, glutathione S transferase and lysozyme.
  • the carboxy-te ⁇ ninus of an intein can be fused with a specific chitin-binding domain. This binds tightly to a packed column of chitin beads and can be used for the affinity-purification of the intact fusion protein. After extensive washing, the column can then be treated with an appropriate cleavage reagent and the liberated target peptide eluted.
  • any expression system which can operate on a commercial scale is suitable although the intein based vector described above is designed for use in E. coli.
  • Other vectors can be designed for optimal use in a particular expression system. For example, if a mammalian expression system was chosen, then protein-encoding regions should have optimised codon usage for that particular system. Expression could also be improved by use of a smaller affinity tag for identification and/or purification such as a repeat of acidic or basic amino acids as described above, to permit resolution from contaminating proteins by ion-exchange chromatography or by the inclusion of a poly-histidine sequence for purification on a metal chelate matrix. A further modification which could improve secretion from a mammalian system (the current E.
  • coli vector is designed for intracellular protein production) would be to add a secretory leader sequence to the calcitonin to promote secretion into the media or into the milk of transgenic animals.
  • a secretory leader sequence to be added to the calcitonin to promote secretion into the media or into the milk of transgenic animals.
  • a leader sequence should be removed during the secretory process by natural processing enzymes.
  • Examples of expression systems which could be used to express peptide fusion proteins include bacteria (E.coli, B.subtilis etc.), yeast (S. cerevisiae, P.pastoralis etc.), insect cells (S. frugiperda), mammalian expression systems (Chinese hamster ovary, baby hamster kidney etc.), transgenic mammalian expression in milk or other body fluids (preferably pig, cow, sheep, goat, rabbit etc) and plants (potato, corn, etc).
  • E.coli expression system the initiator methionine will be retained in the expression product.
  • this initiator methionine can be removed using cyanogen bromide.
  • cyanogen bromide One example of such a peptide is Calcitonin.
  • Expression could be optimised for any of these systems, and for intracellular or extracellular production, by the appropriate selection of leader sequence, codon usage, intein or mutant thereof, and purification strategy.
  • this invention is not tied to any particular manifestation of intein or any species as a source. For instance, it may not be necessary to use a whole intein molecule, much of the sequence may be irrelevant to the desired process and perhaps most of the molecule is functionally unnecessary. Indeed, other proteins outside the definition of "intein” may be capable of transferring the peptide bond at the carboxy-terminus of the target peptide to an appropriate thiol group thus creating the thio-ester group which is necessary for cleavage with concomitant amidation.
  • Thio-esters are relatively reactive, chemical groups-, compared to either peptide bonds or oxygen-esters, and are therefore readily converted to amides under mild reactive conditions. There are two points in the normal cleavage and release pathway during which the fused peptide can be converted to a carboxy-terminal amide. The first and probably most suitable point is after the peptide has been released from the fusion partner by the addition of a thiol reagent.
  • the preferred reagent is dithiothreitol but any number of sulphur-containing reductants could also function effectively.
  • This reaction is essentially a thiol-interchange reaction where the thiol-ester formed between the carboxy-terminus of the peptide and the sulphur of the intein cysteine is transferred to one of the dithiothreitol sulphur atoms.
  • the acyl shift reaction between the amine of the cysteine at the an o-terminus of the intein and the sulphur of the same amino acid residue, is an equilibrium. With the yeast intein described above this equilibrium is shifted in favour of the amine group and the thio-ester is a minor component.
  • the added thiol reagent removes this thio-ester species and therefore drives the reaction in the direction of making more thio-ester until effectively all of the peptide is released as free thio-ester.
  • the released thio-ester is relatively stable to hydrolysis by water (which would generate the unwanted free acid) and is thus suitable for cleavage by any chemical conditions which will promote amide formation.
  • the second point where the peptide exhibits a thio-ester is to the intein itself but as described above, this species is a minor component. However, even here it would be possible to design chemical conditions to allow simultaneous release of the peptide as an amidated species.
  • amides can be formed by the cleavage of thio-esters with ammonia and related compounds. This requires conditions where the positive charge of the carbonyl is enhanced (which is an effect of the adjacent sulphur atom) and the lone pair of electrons on the nitrogen of ammonia are available.
  • the positively-charged ammonium ion provided by a salt such as ammonium phosphate or sulphate, is in equilibrium with uncharged ammonia, the reactive species, and the concentration of free ammonia is thus increased with a lowering of the hydrogen ion concentration. It is therefore expected that the reaction promoting the formation of the amide product, although likely to proceed at relatively low pH values, for example pH 4.0 to 6.0, will occur more rapidly as the pH is increased in the range 6.0 to 9.0 or even 10.0, where the equilibrium is shifted significantly in favour of ammonia formation.
  • a salt such as ammonium phosphate or sulphate
  • the optimal range will be a compromise between the highest pH which will be tolerated by the peptide substrate itself and the lowest pH whereby the reaction still proceeds at an acceptable rate.
  • This optimum range will be deterrnined by the sequence of the peptide itself and other factors relating to the properties of the fusion partner and to process-related, especially purification, issues. Similar conditions and constraints are likely to apply whether the cleavage/amidation reactions occur simultaneously or sequentially.
  • Vector pCYBl obtainable from New England Biolabs, containing a Ndel site for translation initiation and a Sapl site directly adjacent to the intein, was used to clone and express glycine extended salmon calcitonin (sCT-G).
  • the sCT-G coding sequence was synthesised as two complementary single stranded oligonucleotides of 103bases and 104bases. The codon usage was optimised for expression in E.coli. Annealing of the two strands produced 5' overhangs complementary to the Ndel (5' end) and the Sapl site (3' end).
  • the double stranded oligonucleotide was inserted into pCYBl digested with Ndel and Sapl.
  • the expression of the fusion gene is under the control of the P ⁇ promoter and is regulated by IPTG due to the presence of a lacl q gene on the vector.
  • the pCYBl vector containing sCT-G was transfected into DH5- ⁇ , cells grown, induced with IPTG, harvested and lysed by sonication. Expressed fusion was captured on chitin agarose which was washed and then boiled in SDS-PAGE sample buffer. The supernatant was run on 16% SDS-PAGE gels and the protein visualised with coomassie stain or electroblotted to PVDF membrane for N- terminal sequencing. The sequence analysis indicated that the sCT-G was N- terminally truncated at two positions; Ser2 and Thr6. 1.3. Fusion Protein Cleavage and Peptide Amidation
  • Chitin agarose bound fusion was washed with 20mM Hepes pH 8.0, 40mM DTT (cleavage buffer A) or with cleavage buffer A supplemented with 3.0M ammonium bicarbonate (cleavage buffer B) and incubated at 4°C overnight. Released sCT-G was washed from the column and captured on a cation exchange resin then eluted with a salt step.
  • the LHRH fusion was treated in the same manner as the sCT-G fusion until the final cation capture step.
  • the column wash was applied directly to an elecrospray mass spectrometer and the data reconstructed to give the mass of the parent ion ( Figure 2).
  • LHRH from cleavage buffer B (as described in example 1) resulted in a parent ion with a mass of 133 IDa consitent with the Met extended, amidated molecule.
  • LHRH from cleavage buffer A (as described in example 1) gave a parent ion mass of 1332 Da consistent with the Met extended free acid.
  • the difference of IDa is the expected mass difference between an amide and carboxylic acid.
  • the IMPACT I Intein Mediated Purification with an Affinity Chitin-binding Tag protein purification system from New England Biolabs (NEB) offers 4 E. coli expression vectors, which differ in their available cloning sites.
  • Human Amylin is cloned using the NEB vector pCYBl, which contains a Ndel site for translation initiation and a Sapl site directly adjacent to the intein.
  • the Human Amylin sequence is synthesised as two complementary single stranded oligo nucleotides of 115 and 116 bases respectively.
  • the codon usage is optimised for expression in E. coli. Annealing of the two strands produces 5' overhangs complementary to the Ndel (5' end) and the Sapl site (3' end).
  • the double stranded oligo nucleotide can be inserted directly into pCYBl which has previously been digested with both Ndel and Sapl.
  • Expression in bacteria requires transformation of cells with an expression construct using any one of a range of standard methods (Maniatis et al, supra). After cell growth, it is usual to induce expression of the target fusion protein using a combination of an inducible promoter, for example the ⁇ -galactosidase promoter, and a small molecule inducer such as IPTG.
  • the fusion protein is then recovered after cell harvesting and breakage and then purified by affinity chromatography. Most usually, this involves passing the clarified cell lysate through a column of an appropriate affinity matrix displaying a ligand to which the fusion protein binds. Contaminants are then washed from the matrix before either specific elution of the fusion protein or cleavage of the bound fusion protein in situ.
  • the fusion protein containing lysozyme would be purified by cation exchange chromatography.
  • cleavage in situ is probably not an option, unless cleavage conditions can be found which do not promote elution of the fusion protein. Under these circumstances, cleavage in solution phase would be required. Cleavage of the fusion protein whilst bound to a matrix simplifies the subsequent purification of the peptide.
  • Cleavage of the fusion protein can be done by the direct addition of a thiol acyl- acceptor, such as lOmM DTT, to yield a thioester intermediate, which can subsequently be converted to the amide by treatment with ammonia salts at a pH above 6.0. Simultaneous cleavage and conversion t an amide may also be possible with the addition of a suitable mixture of acceptor thiol and ammonia salt.
  • a thiol acyl- acceptor such as lOmM DTT
  • Released peptide is then further purified, if necessary, using conventional techniques such as solvent partitioning and HPLC.

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Abstract

L'invention concerne des méthodes de production de peptides, en particulier mais de manière non exhaustive avec des modifications de l'extrémité carboxylique telles qu'une amidation, par des techniques de recombinaison.
PCT/GB1998/001281 1997-05-01 1998-05-01 Methodes de production d'un peptide amide par utilisation d'une proteine de fusion WO1998050563A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002287204A CA2287204A1 (fr) 1997-05-01 1998-05-01 Methodes de production d'un peptide amide par utilisation d'une proteine de fusion
EP98919369A EP0979291A1 (fr) 1997-05-01 1998-05-01 Methodes de production d'un peptide amide par utilisation d'une proteine de fusion
JP54783598A JP2001525664A (ja) 1997-05-01 1998-05-01 融合タンパク質を使用するアミド化されたペプチドの製造方法
KR19997010114A KR20010012165A (ko) 1997-05-01 1998-05-01 융합 단백질을 사용하여 아미드화 펩티드를 생성하는 방법
NZ500507A NZ500507A (en) 1997-05-01 1998-05-01 Production of an amidated peptide through the use of a fusion protein and release from the fusion protein using an acyl-acceptor
AU72244/98A AU7224498A (en) 1997-05-01 1998-05-01 Methods of production of an amidated peptide through the use of a fusion protein

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GBGB9708918.9A GB9708918D0 (en) 1997-05-01 1997-05-01 Methods
GB9708918.9 1997-05-01

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CN (1) CN1254379A (fr)
AU (1) AU7224498A (fr)
CA (1) CA2287204A1 (fr)
GB (1) GB9708918D0 (fr)
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032518A1 (fr) * 1997-12-19 1999-07-01 Hormos Medical Oy Ltd. Molecule d'adn codant un prepro-neuropeptide y mutant, peptide signal mutant et ses utilisations
WO2001012820A1 (fr) * 1999-08-17 2001-02-22 Health Research Institute Systeme genetique et inteines d'autoclivage derivees, bioseparations et purification de proteines utilisant ces inteines, et procede de determination des restes d'acides amines critiques, generalisables pour modifier l'activite des inteines
US6312898B1 (en) 1999-04-15 2001-11-06 Hormos Medical Oy, Ltd. Diagnosis of a person's risk of developing atherosclerosis or diabetic retinopathy based on leucine 7 to proline 7 polymorphism in the prepro-neuropeptide Y gene
EP1237900A1 (fr) * 1999-09-17 2002-09-11 Genzyme Transgenics Corporation Proteine de fusion optimisee par des sous-unites
WO2006132925A2 (fr) * 2005-06-01 2006-12-14 University Of Pittsburgh Of The Commonwealth System Of Higher Education Procede de biosynthese de peptide amide et administration d'endomorphine-2 in vivo en vue du traitement de la douleur
US7582289B2 (en) 1999-11-12 2009-09-01 Oncolytics Biotech Inc. Viruses for the treatment of cellular proliferative disorders
WO2010028122A1 (fr) 2008-09-03 2010-03-11 Scinopharm Taiwan Ltd. Procédé de fabrication de bivalirudine
WO2010075983A1 (fr) 2008-12-29 2010-07-08 Lonza Braine Sa Procédé pour la production de bivalirudine
WO2016130899A1 (fr) 2015-02-13 2016-08-18 The Board Of Trustees Of The University Of Illinois Inhibition peptidique des maladies ou affections médiées par le ccr3
US9670257B2 (en) 2013-05-31 2017-06-06 Novo Nordisk A/S Methods for producing peptides using engineered inteins
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
WO2021021774A1 (fr) 2019-07-29 2021-02-04 The Board Of Trustees Of The University Of Illinois Composition et méthode pour favoriser la cicatrisation des plaies

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JP4934397B2 (ja) * 2006-10-19 2012-05-16 学校法人順天堂 トランスジェニック非ヒト動物
KR102000490B1 (ko) * 2018-01-12 2019-10-01 전남대학교산학협력단 가용성이 개선된 살모넬라균 편모 유래 플라젤린 단백질 발현 형질전환체, 그 제조방법 및 용도

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Cited By (21)

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Publication number Priority date Publication date Assignee Title
US6046317A (en) * 1997-12-19 2000-04-04 Hormos Medical Oy, Ltd. DNA molecule encoding a mutant prepro-neuropeptide Y, a mutant signal peptide, and uses thereof
US7084242B2 (en) 1997-12-19 2006-08-01 Hormos Medical Oy Ltd. DNA molecule encoding a mutant prepro-neuropeptide Y, a mutant signal peptide, and uses thereof
WO1999032518A1 (fr) * 1997-12-19 1999-07-01 Hormos Medical Oy Ltd. Molecule d'adn codant un prepro-neuropeptide y mutant, peptide signal mutant et ses utilisations
US6312898B1 (en) 1999-04-15 2001-11-06 Hormos Medical Oy, Ltd. Diagnosis of a person's risk of developing atherosclerosis or diabetic retinopathy based on leucine 7 to proline 7 polymorphism in the prepro-neuropeptide Y gene
WO2001012820A1 (fr) * 1999-08-17 2001-02-22 Health Research Institute Systeme genetique et inteines d'autoclivage derivees, bioseparations et purification de proteines utilisant ces inteines, et procede de determination des restes d'acides amines critiques, generalisables pour modifier l'activite des inteines
EP1237900A1 (fr) * 1999-09-17 2002-09-11 Genzyme Transgenics Corporation Proteine de fusion optimisee par des sous-unites
EP1237900A4 (fr) * 1999-09-17 2005-08-03 Gtc Biotherapeutics Inc Proteine de fusion optimisee par des sous-unites
US7582289B2 (en) 1999-11-12 2009-09-01 Oncolytics Biotech Inc. Viruses for the treatment of cellular proliferative disorders
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
US8846889B2 (en) 2005-06-01 2014-09-30 Darren P. Wolfe Peptide biosynthesis and pain therapy
US7825231B2 (en) 2005-06-01 2010-11-02 Darren P. Wolfe Method of amidated peptide biosynthesis and delivery in vivo: endomorphin-2 for pain therapy
US8003622B2 (en) 2005-06-01 2011-08-23 Darren Wolfe Peptide biosynthesis and pain therapy
WO2006132925A3 (fr) * 2005-06-01 2007-03-15 Univ Pittsburgh Procede de biosynthese de peptide amide et administration d'endomorphine-2 in vivo en vue du traitement de la douleur
WO2006132925A2 (fr) * 2005-06-01 2006-12-14 University Of Pittsburgh Of The Commonwealth System Of Higher Education Procede de biosynthese de peptide amide et administration d'endomorphine-2 in vivo en vue du traitement de la douleur
WO2010028122A1 (fr) 2008-09-03 2010-03-11 Scinopharm Taiwan Ltd. Procédé de fabrication de bivalirudine
US8252896B2 (en) 2008-09-03 2012-08-28 ScnioPharm Taiwan, Ltd. Process for making bivalirudin
WO2010075983A1 (fr) 2008-12-29 2010-07-08 Lonza Braine Sa Procédé pour la production de bivalirudine
US8921517B2 (en) 2008-12-29 2014-12-30 Lonza Braine Sa Process for the production of bivalirudin
US9670257B2 (en) 2013-05-31 2017-06-06 Novo Nordisk A/S Methods for producing peptides using engineered inteins
WO2016130899A1 (fr) 2015-02-13 2016-08-18 The Board Of Trustees Of The University Of Illinois Inhibition peptidique des maladies ou affections médiées par le ccr3
WO2021021774A1 (fr) 2019-07-29 2021-02-04 The Board Of Trustees Of The University Of Illinois Composition et méthode pour favoriser la cicatrisation des plaies

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JP2001525664A (ja) 2001-12-11
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KR20010012165A (ko) 2001-02-15
EP0979291A1 (fr) 2000-02-16
CN1254379A (zh) 2000-05-24
AU7224498A (en) 1998-11-27
CA2287204A1 (fr) 1998-11-12

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