RU2218407C2 - Прямая экспрессия пептидов в культуральные среды - Google Patents
Прямая экспрессия пептидов в культуральные среды Download PDFInfo
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- RU2218407C2 RU2218407C2 RU99124196/13A RU99124196A RU2218407C2 RU 2218407 C2 RU2218407 C2 RU 2218407C2 RU 99124196/13 A RU99124196/13 A RU 99124196/13A RU 99124196 A RU99124196 A RU 99124196A RU 2218407 C2 RU2218407 C2 RU 2218407C2
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract 19
- 102000004196 processed proteins & peptides Human genes 0.000 title 1
- 239000013598 vector Substances 0.000 claims abstract 13
- 230000001105 regulatory effect Effects 0.000 claims abstract 8
- 108020004707 nucleic acids Proteins 0.000 claims abstract 7
- 150000007523 nucleic acids Chemical class 0.000 claims abstract 7
- 102000039446 nucleic acids Human genes 0.000 claims abstract 7
- 238000012258 culturing Methods 0.000 claims abstract 5
- 241000588724 Escherichia coli Species 0.000 claims abstract 4
- 210000004027 cell Anatomy 0.000 claims abstract 4
- 238000000034 method Methods 0.000 claims abstract 4
- 230000002708 enhancing effect Effects 0.000 claims abstract 3
- 230000028327 secretion Effects 0.000 claims abstract 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract 2
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract 2
- 229910052799 carbon Inorganic materials 0.000 claims abstract 2
- 239000002609 medium Substances 0.000 claims 5
- 239000001963 growth medium Substances 0.000 claims 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims 3
- 108091026890 Coding region Proteins 0.000 claims 2
- 239000004471 Glycine Substances 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims 1
- 108700026244 Open Reading Frames Proteins 0.000 claims 1
- 238000005277 cation exchange chromatography Methods 0.000 claims 1
- 239000013613 expression plasmid Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 239000012528 membrane Substances 0.000 claims 1
- 230000035699 permeability Effects 0.000 claims 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 1
- 230000002103 transcriptional effect Effects 0.000 claims 1
- 239000013600 plasmid vector Substances 0.000 abstract 2
- 230000004071 biological effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 239000012530 fluid Substances 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 210000003705 ribosome Anatomy 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/23—Calcitonins
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- 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
- C07K14/585—Calcitonins
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- 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
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
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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
- C12N15/72—Expression systems using regulatory sequences derived from the lac-operon
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- 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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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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Abstract
Изобретение относится к генетической инженерии. Экспрессирующий плазмидный вектор содержит нуклеиновые кислоты (НК), кодирующие пептидный продукт. Указанные НК присоединены в рамке считывания справа (3') от нуклеиновых кислот, кодирующих сигнальный пептид. Регуляторный район оперативно соединен с кодирующим районом. Регуляторный район содержит множество промоторов и хотя бы один сайт связывания рибосом. По меньшей мере один из промоторов представляет собой tac. Вектор может содержать НК, кодирующие, по меньшей мере, один усиливающий секрецию пептид. Культивируют Е.соli, трансформированную или трансфецированную заявленным экспрессирующим плазмидным вектором. Пептид извлекают из среды. Культивирование проводят в присутствии в среде источника углерода и при регулировании роста клеток-хозяев при скорости роста 0,05 и 0,20 удвоений в час. Изобретение позволяет осуществлять прямую экспрессию пептида в культуральную среду за пределами клетки. 4 с. и 9 з.п.ф-лы, 17 ил., 5 табл.
Description
Текст описания в факсимильном виде (см. графическую часть).
Claims (13)
1. Экспрессирующий плазмидный вектор, содержащий (a) кодирующий район с нуклеиновыми кислотами, кодирующими пептидный продукт и присоединенными в рамке считывания справа (3') от нуклеиновых кислот, кодирующих сигнальный пептид; (b) регуляторный район, оперативно соединенный с кодирующим районом, причем указанный регуляторный район содержит множество промоторов и по меньшей мере один сайт связывания рибосом, причем по меньшей мере один из упомянутых промоторов представляет собой tac.
2. Вектор по п.1, отличающийся тем, что содержит множество транскрипционных кассет, причем каждая кассета имеет упомянутый регуляторный район и указанный кодирующий район.
3. Вектор по п.1, отличающийся тем, что указанный регуляторный район имеет точно два промотора.
4. Вектор по п.1, отличающийся тем, что указанный промотор tac находится слева (5') от другого промотора в указанном регуляторном районе.
5. Вектор по п.1, отличающийся тем, что С-концевая аминокислота указанного пептидного продукта представляет собой глицин.
6. Вектор по п.1, отличающийся тем, что дополнительно содержит нуклеиновые кислоты, кодирующие по меньшей мере один усиливающий секрецию пептид.
7. Вектор по п.6, отличающийся тем, что усиливающий секрецию пептид выбран из группы, состоящей из secY и pr1A-4.
8. Вектор по п.1, отличающийся тем, что является введенным в клетку-хозяина.
9. Способ получения пептидного продукта, предусматривающий культивирование Е.соli, трансформированной или трансфецированной вектором по п.1, в культуральной среде и извлечение этого пептидного продукта из среды.
10. Способ получения пептидного продукта, предусматривающий культивирование E.coli, трансформированной или трансфецированной вектором по п.6, в культуральной среде и извлечение этого пептидного продукта из среды.
11. Способ получения пептидного продукта, предусматривающий стадии
(a) культивирования E.coli, трансформированной или трансфецированной вектором по п.1, в культуральной среде в присутствии источника углерода и индукции экспрессии пептидного продукта при регулировании роста этих клеток-хозяев при скорости роста между 0,05 и 0,20 удвоений в час; (b) извлечения после этого упомянутого пептидного продукта из этой среды.
12. Способ по п.11, отличающийся тем, что в среде присутствует увеличивающее проницаемость мембраны количество глицина во время по меньшей мере части периода упомянутого регулируемого роста.
13. Способ по п.11, отличающийся тем, что извлечение упомянутого продукта предусматривает (a) отделение клеток-хозяев от культуральной среды; (b) проведение жидкостной хроматографии с обращенной фазой этой среды и извлечение фракций, содержащих пептидный продукт; (c) проведение катионообменной хроматографии фракций стадии (b) и
(d) извлечение после этого фракций, содержащих пептидный продукт.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US4370097P | 1997-04-16 | 1997-04-16 | |
US60/043,700 | 1997-04-16 |
Publications (2)
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RU99124196A RU99124196A (ru) | 2001-09-20 |
RU2218407C2 true RU2218407C2 (ru) | 2003-12-10 |
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RU99124196/13A RU2218407C2 (ru) | 1997-04-16 | 1998-04-15 | Прямая экспрессия пептидов в культуральные среды |
Country Status (14)
Country | Link |
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US (5) | US6103495A (ru) |
EP (2) | EP1005526B1 (ru) |
JP (2) | JP4907751B2 (ru) |
AT (2) | ATE484572T1 (ru) |
AU (1) | AU742270B2 (ru) |
CA (1) | CA2285658C (ru) |
DE (1) | DE69841939D1 (ru) |
DK (2) | DK1005526T3 (ru) |
ES (1) | ES2375330T3 (ru) |
HU (1) | HUP0001272A3 (ru) |
LT (1) | LT4701B (ru) |
NO (1) | NO327325B1 (ru) |
RU (1) | RU2218407C2 (ru) |
WO (1) | WO1998046722A1 (ru) |
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1998
- 1998-04-15 DK DK98918334.8T patent/DK1005526T3/da active
- 1998-04-15 RU RU99124196/13A patent/RU2218407C2/ru active
- 1998-04-15 DK DK09006784.4T patent/DK2088188T3/da active
- 1998-04-15 HU HU0001272A patent/HUP0001272A3/hu unknown
- 1998-04-15 JP JP54430898A patent/JP4907751B2/ja not_active Expired - Lifetime
- 1998-04-15 AU AU71279/98A patent/AU742270B2/en not_active Ceased
- 1998-04-15 AT AT98918334T patent/ATE484572T1/de not_active IP Right Cessation
- 1998-04-15 US US09/060,765 patent/US6103495A/en not_active Expired - Lifetime
- 1998-04-15 CA CA2285658A patent/CA2285658C/en not_active Expired - Fee Related
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- 1998-04-15 DE DE69841939T patent/DE69841939D1/de not_active Expired - Lifetime
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- 1998-04-15 WO PCT/US1998/007723 patent/WO1998046722A1/en active IP Right Grant
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1999
- 1999-10-14 NO NO19995014A patent/NO327325B1/no not_active IP Right Cessation
- 1999-11-10 LT LT99-133A patent/LT4701B/lt not_active IP Right Cessation
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2000
- 2000-02-08 US US09/500,009 patent/US6210925B1/en not_active Expired - Lifetime
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2001
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- 2001-02-09 US US09/780,643 patent/US6737250B2/en not_active Expired - Lifetime
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2004
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009048354A1 (en) * | 2007-10-08 | 2009-04-16 | Federalnoe Gosudarstvennoe Unitarnoe Predpriyatie 'gosudarstvenny Nauchny Tsentr 'nauchno-Issledovatelsky Institut Organicheskikh Poluproduktov I Krasitelei (Niopik) | Method for overproducing anti-her2/neu oncogene antibodies in plant |
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