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WO1991005052A1 - Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae - Google Patents

Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae Download PDF

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Publication number
WO1991005052A1
WO1991005052A1 PCT/SU1989/000257 SU8900257W WO9105052A1 WO 1991005052 A1 WO1991005052 A1 WO 1991005052A1 SU 8900257 W SU8900257 W SU 8900257W WO 9105052 A1 WO9105052 A1 WO 9105052A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmid
interleukin
activity
gene
δηκ
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.)
Ceased
Application number
PCT/SU1989/000257
Other languages
English (en)
Russian (ru)
Inventor
Andrei Novomirovich Myasnikov
Mikhail Nikolaevich Smirnov
Kirill Veniaminovich Ostanin
Andris Yanovich Avot
Elmar Yanovich Gren
Nadezhda Viktorovna Romanchikova
Alexandr Jurievich Tsimanis
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.)
LENINGRADSKY GOSUDARSTVENNY UNIVERSITET
Original Assignee
LENINGRADSKY GOSUDARSTVENNY UNIVERSITET
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 LENINGRADSKY GOSUDARSTVENNY UNIVERSITET filed Critical LENINGRADSKY GOSUDARSTVENNY UNIVERSITET
Priority to PCT/SU1989/000257 priority Critical patent/WO1991005052A1/fr
Publication of WO1991005052A1 publication Critical patent/WO1991005052A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C12N15/80—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

Definitions

  • Interleukin-2 is related to a group of proteins called lymphokines. These squirrels play a key role in the regulation of the immune system of the body and are considered extremely emergency.
  • the radiopharmaceutical is used for the preparation of purified preparations of Interleukin-2 and is used as a non-food source.
  • interleukin-2 the intracellular synthesis of interleukin-2 is in both the car battery, and in the case of other car cords, it is inappropriate to consume the device present in the native interleukin-2.
  • a dangerous method of eliminating the listed disadvantages is the creation of strains
  • the task posed is achieved by improving the design of a recombinant plasmid that ensures biosynthesis
  • An essential essential condition for solving the posed problem is the inclusion in the composition of the recombinant plasmid of elements that ensure its accumulation in
  • 30 useful plasmids are the strains of the other are strains that are segregated that carry a non-neurological mutation in the L gene.
  • ⁇ ig. 1 Illustrates the structure of the recombinant plasmid ⁇ ( ⁇ -), which ensures the biosynthesis and secretion of interleukin-2 from the cells of the friend.
  • ⁇ ig. 3. Illustrates the ways of the plasmid ⁇ (LB ⁇ ) conversion.
  • the plasmid RL5 ⁇ ( ⁇ - ⁇ P-,), the conversion will allow you to receive another strain with the listed properties, consists of the following 20 elements:
  • - Community 47 is a gene fragment of the other, containing part of the territory and the risk of the participation of this gene, ⁇ measurement 0.45 tl;
  • FIG. 1 10 plasmid structure is illustrated in FIG. 1.
  • This fragment is a one-day league with 0.9 t, ⁇ . ⁇ - ⁇ ⁇ agment ⁇ of the plasmid ⁇ [Explained ⁇ - ⁇ . and d ⁇ . Resolved. application No. 4392922/13 (1988)] and the majority of the plasmid RL) ⁇ 207. Obtained by the aforementioned method, a definitive genetically engineered structure (plasmid RL (S) (LNP.)), Are merged into
  • Bacteria v. SOSI containing plasmid ⁇ 69 ⁇ , grow during the night in 500 ml of nutrient medium B Wu (1 prim., 03
  • the unit adds 0.6 volume of the product and separates the plant with a low centrifuge (5000 rpm) at 4 deg. It is dried in a vacuum, dispersed in 33 ml of water, added 5 g of cesium solution and 100 ml of a solution of etidium bromide (mg / ml) and
  • s ⁇ de ⁇ z haschy ⁇ m ⁇ E ⁇ and 1 mg / l of bromide etidium).
  • Particularly sensitive area ⁇ with a size of 1.6 t.o. cut a hole (3x60 mm) and place a dialysis membrane of the corresponding size into this hole 25 and fill it with a buffer. Electricity lasts for another 10-15 minutes, after which a buffer from the hole is removed, one is added to the battery and one or two more are added, it is added that it is added.
  • ⁇ Separate the centrifuge at 10,000 and 30 rpm for 10 minutes, wash the plant with ethanol, dry it in a vacuum and dispense 100 ml of water.
  • linear linear plasmid form is similar to that described above for the ⁇ plasmid ⁇ 69 ⁇ fragment.
  • the bank uses the processing unit.
  • nucleic acid precipitate obtained by precipitating from the panel, it grows in 100 microliters of buffer for testing and
  • ⁇ 10 treats the following combinations of experimental endonucleases ( ⁇ 10 units): ⁇ + ⁇ (the required plasmid yields fragments 1,1, 03 t.o.) and ⁇ locker ⁇ ( ⁇ motivationagment) 1,6 ⁇ . From the conventional method such as the transverse clone, which contains the plasmid ⁇ ( ⁇ ) -2, the receptive plasmid secrete plasmid ⁇ réelle, as for ⁇ plasmid.
  • the plasmid ⁇ oul ⁇ ( ⁇ ) -2 ( ⁇ g) is hydrolyzed with a mixture ( ⁇ 100 units) of ⁇ a ⁇ and ⁇ in 100 ⁇ l of ⁇ buffer for 3 hours.
  • Particle ⁇ with a size of 1.1 T. o. Cleans the elec- trophoresis in an agar gel, as described above. By the methods of the drug, they clean the veterinary ⁇ réelle ⁇ -
  • the plasmid vector ⁇ 137 is operated with the use of the above methods for the following scheme: from the plasmid ⁇ , the hydrolyzed ⁇ at ⁇ , isolates 0.6. ⁇ agment
  • Phase ⁇ which contains the coding part of the gene of interleukin-2, is isolated from the hydrolyzed processed product of plasmid ⁇ 12.13- 23 and of the processed product (1).
  • the structure of the plasmid rL ⁇ (LI ⁇ ) (Fig. 1) confirms the decompressive analysis using the decomposition enzyme, ⁇ , ⁇ and ⁇ , as well as the other.
  • burnt eggs which is illustrated by the following example.
  • the batteries are twice washed with water and one at a time for a sodium-citric buffer containing 1 ⁇ of the output, they are suspended at the same time and processed for 150 minutes at a time. ⁇ and 30 grams of ⁇ , and then 150 ⁇ l of glucurinidase within 30-
  • test methods are divided up to 15: 1 50 mb
  • the concentration of interleukin-2 in the analyzed products is determined by comparing the value of absorption in the experimental samples with the experiment in the same way.
  • the cells of the other strain 18, which are transformed with the plasmid rP), are secured in

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention a trait à la biotechnologie et, notamment, à des procédés de génie génétique permettant d'obtenir des protéines recombinées dans des cellules de levure. L'objet de l'invention est de créer une souche de levure, Saccharomyces cerevisiae, capable d'une synthèse et d'une secrétion efficaces d'un polypeptide présentant une activité d'interleukine-2 humaine, ainsi que la régulation efficace telle que la synthèse par variation des conditions permettant la culture de la souche, ce qui est nécessaire pour maintenir stable ladite souche et pour permettre sa culture industrielle. On a mis au point un procédé d'obtention de producteurs de souches de levure, secrétant un polypeptide présentant une activité d'interleukine-2 humaine, une structure ainsi qu'un procédé d'élaboration d'un plasmide recombiné assurant la synthèse et la secrétion d'interleukine-2. On parvient à une stabilité et à une productivité accrue de ces souches, par comparaison avec celles connues à ce jour, par l'emploi, dans l'élaboration du plasmide, d'un promoteur de levure hybride. Les cellules de souches de levure porteuses desdits plasmides, secrètent dans un milieu de culture, une protéine ayant l'activité de l'interleukine-2 humaine, en une quantité d'environ quelques milligrammes par litre de culture de levure. L'activité biologique spécifique de ladite protéine est pratiquement égale à celle de l'interleukine-2 naturelle.
PCT/SU1989/000257 1989-09-28 1989-09-28 Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae Ceased WO1991005052A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/SU1989/000257 WO1991005052A1 (fr) 1989-09-28 1989-09-28 Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/SU1989/000257 WO1991005052A1 (fr) 1989-09-28 1989-09-28 Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae

Publications (1)

Publication Number Publication Date
WO1991005052A1 true WO1991005052A1 (fr) 1991-04-18

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SU1989/000257 Ceased WO1991005052A1 (fr) 1989-09-28 1989-09-28 Procede d'obtention d'un polypeptide presentant une activite d'interleukine-2 humaine, secrete par des cellules de levure, saccharomyces cerevisiae

Country Status (1)

Country Link
WO (1) WO1991005052A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0505500B1 (fr) * 1989-12-22 1997-07-30 Applied Research Systems Ars Holding N.V. Modification de l'expression de genes endogenes a l'aide d'un element regulateur par recombinaison homologue
US7056701B2 (en) 1992-01-31 2006-06-06 Aventis Behring L.L.C. Hormone and albumin fusion protein
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
US7507414B2 (en) 2000-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins
KR101628605B1 (ko) 2015-02-03 2016-06-21 현대자동차주식회사 자동차용 와이퍼 장치

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0142268A1 (fr) * 1983-10-18 1985-05-22 Ajinomoto Co., Inc. Saccharomyces cerevisiae possédant le gène codant pour le polypeptide interleukine-2 et procédé de production d'interleukine-2 utilisant la levure
EP0194818A2 (fr) * 1985-03-11 1986-09-17 Takeda Chemical Industries, Ltd. Préparation d'interleukine-2
US4738927A (en) * 1982-03-31 1988-04-19 Ajinomoto Co. Inc. Gene coded for interleukin-2 polypeptide, recombinant DNA carrying the said gene, a living cell line possessing the recombinant DNA, and method for producing interleukin-2 using the said cell

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4738927A (en) * 1982-03-31 1988-04-19 Ajinomoto Co. Inc. Gene coded for interleukin-2 polypeptide, recombinant DNA carrying the said gene, a living cell line possessing the recombinant DNA, and method for producing interleukin-2 using the said cell
EP0142268A1 (fr) * 1983-10-18 1985-05-22 Ajinomoto Co., Inc. Saccharomyces cerevisiae possédant le gène codant pour le polypeptide interleukine-2 et procédé de production d'interleukine-2 utilisant la levure
EP0194818A2 (fr) * 1985-03-11 1986-09-17 Takeda Chemical Industries, Ltd. Préparation d'interleukine-2

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0505500B1 (fr) * 1989-12-22 1997-07-30 Applied Research Systems Ars Holding N.V. Modification de l'expression de genes endogenes a l'aide d'un element regulateur par recombinaison homologue
US7410779B2 (en) 1992-01-31 2008-08-12 Novozymes Biopharma Uk Limited Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
US7056701B2 (en) 1992-01-31 2006-06-06 Aventis Behring L.L.C. Hormone and albumin fusion protein
US7081354B2 (en) 1992-01-31 2006-07-25 Aventis Behring L.L.C. Interleukin and albumin fusion protein
US7507414B2 (en) 2000-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7592010B2 (en) 2001-12-21 2009-09-22 Human Genome Sciences, Inc. Albumin fusion proteins
US7238667B2 (en) 2001-12-21 2007-07-03 Human Genome Sciences, Inc. Albumin fusion proteins
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
US7847079B2 (en) 2001-12-21 2010-12-07 Human Genome Sciences, Inc. Albumin fusion proteins
US8071539B2 (en) 2001-12-21 2011-12-06 Human Genome Sciences, Inc. Albumin fusion proteins
US8252739B2 (en) 2001-12-21 2012-08-28 Human Genome Sciences, Inc. Albumin fusion proteins
US8513189B2 (en) 2001-12-21 2013-08-20 Human Genome Sciences, Inc. Albumin fusion proteins
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins
KR101628605B1 (ko) 2015-02-03 2016-06-21 현대자동차주식회사 자동차용 와이퍼 장치

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