EP0979291A1 - Verfahren zur herstellung von amidierten peptiden durch anwendung von einem fusionsprotein - Google Patents

Verfahren zur herstellung von amidierten peptiden durch anwendung von einem fusionsprotein

Info

Publication number
EP0979291A1
EP0979291A1 EP98919369A EP98919369A EP0979291A1 EP 0979291 A1 EP0979291 A1 EP 0979291A1 EP 98919369 A EP98919369 A EP 98919369A EP 98919369 A EP98919369 A EP 98919369A EP 0979291 A1 EP0979291 A1 EP 0979291A1
Authority
EP
European Patent Office
Prior art keywords
fusion protein
peptide
expressed
sequence
transgenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98919369A
Other languages
English (en)
French (fr)
Inventor
Ian Robert Cottingham
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PPL Therapeutics Scotland Ltd
Original Assignee
PPL Therapeutics Scotland Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PPL Therapeutics Scotland Ltd filed Critical PPL Therapeutics Scotland Ltd
Publication of EP0979291A1 publication Critical patent/EP0979291A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

  • 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.
  • 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.
  • 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.
  • 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 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Environmental Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Textile Engineering (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP98919369A 1997-05-01 1998-05-01 Verfahren zur herstellung von amidierten peptiden durch anwendung von einem fusionsprotein Withdrawn EP0979291A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9708918.9A GB9708918D0 (en) 1997-05-01 1997-05-01 Methods
GB9708918 1997-05-01
PCT/GB1998/001281 WO1998050563A1 (en) 1997-05-01 1998-05-01 Methods of production of an amidated peptide through the use of a fusion protein

Publications (1)

Publication Number Publication Date
EP0979291A1 true EP0979291A1 (de) 2000-02-16

Family

ID=10811692

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98919369A Withdrawn EP0979291A1 (de) 1997-05-01 1998-05-01 Verfahren zur herstellung von amidierten peptiden durch anwendung von einem fusionsprotein

Country Status (9)

Country Link
EP (1) EP0979291A1 (de)
JP (1) JP2001525664A (de)
KR (1) KR20010012165A (de)
CN (1) CN1254379A (de)
AU (1) AU7224498A (de)
CA (1) CA2287204A1 (de)
GB (1) GB9708918D0 (de)
NZ (1) NZ500507A (de)
WO (1) WO1998050563A1 (de)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
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
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 (en) * 1999-08-17 2001-02-22 Health Research Institute Genetic system and self-cleaving inteins derived therefrom, bioseparations and protein purification employing same, and methods for determining critical, generalizable amino acid residues for varying intein activity
HUP0202702A2 (hu) * 1999-09-17 2002-12-28 Genzyme Transgenics Corporation Alegységoptimalizált fúziós fehérjék és alkalmazásai
EP1955703A1 (de) 1999-11-12 2008-08-13 Oncolytics Biotech Inc. Viren für die Behandlung von zellulären, proliferativen Erkrankungen
JP4903709B2 (ja) 2004-10-19 2012-03-28 ロンザ アーゲー 固相ペプチド合成のための方法
WO2006132925A2 (en) 2005-06-01 2006-12-14 University Of Pittsburgh Of The Commonwealth System Of Higher Education Method of amidated peptide biosynthesis and delivery in vivo: endomorphin-2 for pain therapy
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
KR101634830B1 (ko) 2008-09-03 2016-06-29 시노팜 타이완 리미티드 비발리루딘의 제조방법
SG171859A1 (en) 2008-12-29 2011-07-28 Lonza Braine Sa Process for the production of bivalirudin
WO2014191455A1 (en) 2013-05-31 2014-12-04 Novo Nordisk A/S Methods for producing peptides using engineered inteins
EP3256144B1 (de) 2015-02-13 2021-03-24 The Board of Trustees of the University of Illionis Peptidhemmung von ccr3-vermittelten krankheiten oder zuständen
KR102000490B1 (ko) * 2018-01-12 2019-10-01 전남대학교산학협력단 가용성이 개선된 살모넬라균 편모 유래 플라젤린 단백질 발현 형질전환체, 그 제조방법 및 용도
US20220265759A1 (en) 2019-07-29 2022-08-25 The Board Of Trustees Of The University Of Illiois Composition and method for promoting wound healing

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9127250D0 (en) * 1991-12-23 1992-02-19 Unilever Plc Modification
DK0651761T3 (da) * 1992-07-13 2003-02-10 Bionebraska Inc Fremgangsmåde til modifikation af rekombinante polypeptider
GB9406974D0 (en) * 1994-04-08 1994-06-01 Pharmaceutical Proteins Ltd Transgenic production
JP4476360B2 (ja) * 1995-06-28 2010-06-09 ニユー・イングランド・バイオレイブズ・インコーポレイテツド 改質タンパク質及びその製造法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9850563A1 *

Also Published As

Publication number Publication date
NZ500507A (en) 2001-08-31
KR20010012165A (ko) 2001-02-15
AU7224498A (en) 1998-11-27
WO1998050563A1 (en) 1998-11-12
CN1254379A (zh) 2000-05-24
JP2001525664A (ja) 2001-12-11
CA2287204A1 (en) 1998-11-12
GB9708918D0 (en) 1997-06-25

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