ES2550761T3 - Método para producción de polipéptidos - Google Patents
Método para producción de polipéptidos Download PDFInfo
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- ES2550761T3 ES2550761T3 ES10782288.4T ES10782288T ES2550761T3 ES 2550761 T3 ES2550761 T3 ES 2550761T3 ES 10782288 T ES10782288 T ES 10782288T ES 2550761 T3 ES2550761 T3 ES 2550761T3
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- 239000008103 glucose Substances 0.000 description 1
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- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
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- 230000035772 mutation Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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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
- C07K14/575—Hormones
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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
- C12N15/63—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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- 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)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
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- 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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Abstract
Método para producción de un polipéptido amidado C-terminal en el cual el método comprende los pasos siguientes a) cultivo de una cepa de levadura que tiene un gen KEX1 no-funcional y que comprende una secuencia de DNA que codifica un polipéptido con un Gly C-terminal en condiciones para expresión del polipéptido en la cepa de levadura, b) α-amidación in vitro del polipéptido expresado del paso a) o directamente en la célula de producción si la célula tiene la maquinaria necesaria para α-amidación in vivo y c) aislamiento y purificación del péptido amidado en el terminal C.
Description
E10782288
21-10-2015
El fragmento trp1-ΔFA se compone de un fragmento de 476 pares de bases de la región 5' UTR de KEX1, fusionado directamente a un fragmento de 525 pares de bases de la región 3' UTR de KEX1, evitando así el solapamiento de la secuencia homóloga de DNA a la casete TRP1-FA utilizada como marcador seleccionable en el plásmido pFA6a-TRP1 (Longtine, M.S., McKenzie, A., Demarini, D., Shah, N.G., Wach, A., Brachat, A., Philippsen, P. and Pringle,
5 J.R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast: 14., (953-961)). El fragmento PCR de trp1-ΔFA se clonó al vector TOPO-CR2.1 como se describe por el fabricante (Invitrogen), dando como resultado el plásmido pFI379.
Se obtuvo una cepa de levadura de deleción de trp1-ΔFA como sigue:
10 El método utilizado para seleccionar la interrupción del gen TRP1 implica un procedimiento de contraselección. Se cultivóen medios que contenían ácido 5-fluoroantranílico da como resultado anti-metabolismo por las enzimas en el camino biosintético del triptófano. Las células de levadura, que carecen de las enzimas requeridas para la conversión de ácido antranílico en triptófano son resistentes al ácido 5-fluoroantranílico (Toyn, J.H., Gunyuzlu, P.L.,
15 White, H., Thompson, L.A. and G.F. Hollis (2000). A counterselection for the tryptophan pathway in yeast: 5fluoroanthranilic acid resistance. Yeast: 16 (553-560)).
La cepa hospedadora de levadura NNY574 (MATα ura3-D0 leu2-D0 his3-D0 tpi1::URA3 yps1::HIS3 pep4::LEU2) se transformó con el plásmido pSA281, un vector de expresión de levadura para expresión secretora de un precursor
20 análogo de amilina EEAEK(SEQ ID NO:1)-amilina (1-37)-K1 E, S28P, S29P, G38 para crear yFI754. La cadena principal plasmídica de pSA281 está basada en el vector de tipo cPOT que facilita el crecimiento en medios ricos que contienen glucosa como única fuente de carbono por complementación de la mutación delta-tpi1 de la cepa hospedadora.
25 Un fragmento trp1-ΔFA se amplificó por PCR utilizando pFI379 como molde y oligonucleótidos sintéticos y métodos estándar. El fragmento PCR trp1-ΔFA se utilizó directamente para transformación de yFI754 por el método LiAc (Gietz y Wood, 2002). Después de la transformación, las células de levadura se extendieron sobre agar YEPD y se incubaron durante una noche a 30ºC seguido por replicación en placa en un medio mínimo que contenía ácido 5fluoroantranílico. La selección para las células de levadura que contenían el alelo de deleción trp1-ΔFA integrado en
30 el genoma se realizó por contraselección respecto a la presencia del gen TRP1 utilizando ácido 5-fluoroantranílico (Toyn et al, 2000) dando como resultado la cepa de levadura trp1-ΔFA yFI810.
El alelo de interrupción del gen Δkex1::TRP1-FA se construyó como sigue:
35 Un fragmento de DNA sintético del alelo Δkex1-D0 compuesto de 518 pares de bases de la secuencia de DNA 5'UTR de KEX1 seguido por un sitio de la endonucleasa de restricción SphI, seguido por un sitio de la endonucleasa de restricción Sbf1, seguido por 304 pares de bases de la secuencia de DNA 3'UTR de KEX1 se construyó por síntesis de DNA in vitro a partir de una fuente comercial y se obtuvo clonado en un vector pMA (Geneart AG, BioPark, Josef-Engert-Str. 11, D-93053 Regensburg, Alemania). Este plásmido se designó pFI472. Un fragmento de
40 DNA sintético que contenía la casete TRP1-FA flanqueada por los sitios de las endonucleasas de restricción SphI y Sbf1 se construyó por síntesis de DNA in vitro a partir de una fuente comercial y se obtuvo clonado en un vector pMA-RQ (GeneArt ag). Este plásmido se designó pFI468. Un plásmido, pFI473, que contenía el alelo de interrupción del gen Δkex1::TRP1-FA se construyó por ligación de dos fragmentos de DNA: un fragmento de 3163 pares de bases obtenido después de digestión de pFI472 con SphI y Sbf1 y un fragmento de 872 pares de bases obtenido
45 después de digestión de pFI468 con SphI y Sbf1. Finalmente, se digirió pFI473 con XhoI e HincII y se aisló un fragmento de DNA de 1707 pares de bases que contenía el alelo Δkex1::TRP1-FA y se utilizó para transformación de yFI810 por el procedimiento de LiAc (Gietz, R.D. and Wood, R. (2002). Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method. Methods in Enzymology: 350, (87 -96)). Después de la transformación, las células de levadura se extendieron sobre placas mínimas sin triptófano a fin de seleccionar
50 mutantes con la deleción Δkex1::TRP1-FA. Se caracterizaron transformantes TRP+ por PCR para confirmar la integración correcta del alelo Δkex1::TRP1-FA en el locus KEX1. Se aisló la cepa de levadura yFI815 como un mutante de deleción Δkex1::TRP1-FA (MATα ura3-D0 leu2-D0 his3-D0 trp1-delta-FA tpi1::URA3 yps1::HIS3 pep4::LEU2 kex1::TRP1-FA, pSA281).
Interrupción del gen Δkex1::KanMX4
Una cepa de levadura con el gen KEX1 delecionado y el genotipo Matα his3D1 leu2D0 lys2D0 ura3D0
60 kex1D0::kanMX4 se adquirió de la colección de cepas de deleción Euroscarf (número de acceso Y14570). En esta cepa de levadura, el fragmento de DNA que codifica el marco de lectura abierto de KEX1 ha sido reemplazado con el marcador seleccionable dominante KanMX4, que confiere resistencia al antibiótico G418/geneticina (Wach A., Brachat, A., Pöelmann R. y Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast: 10 (1793-808).
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E10782288
21-10-2015
gaagaa-gctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactattggt (SEQ ID NO:24); gaagaa-gctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactatgggt (SEQ ID NO:25);
5 gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatacttctggt (SEQ ID NO:26); gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactcctggt (SEQ ID NO:27); gaagaa-gctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaatttttt
10 ggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactactggt (SEQ ID NO:28); gaagaa-gctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatacttggggt (SEQ ID NO:29); gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactagaggt (SEQ ID NO:30);
15 gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactcttggt (SEQ ID NO:31); gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaattttttggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactcaaggt (SEQ ID NO:32) y gaagaagctgaaaaagaatgtaatactgctacttgtgctactcaaagattggctaatttttt
20 ggttcattcttctaataattttggtccaattttgccaccaactaatgttggttctaatactaatggt (SEQ ID NO:33).
Se verificó la identidad del vector de expresión, se transformó el plásmido en S.cerevisiae, se cultivó la cepa de levadura, y la proteína secretada se recogió y se analizó como se ha descrito arriba en los ejemplos 3 y 4. Las abundancias de las especies de longitud total y delecionada de glicina se obtuvieron a partir de los espectros
25 simplificados y se utilizaron para estimación del porcentaje de péptido de longitud total frente al conjunto de péptido de longitud total y delecionado de glicina. Los resultados aparecen en la Tabla 2. En la Tabla 2 se ve que el Gly Cterminal en la molécula de amilina se escinde cuando el residuo de aminoácido unido al Gly es uno de Tyr, Phe, Met, Leu, Trp, Ala, Ile y Arg.
30 Tabla 2
- Aminoácido siguiente a Gly (X37)
- % de Longitud total
- Tyr
- 45,5
- Phe
- 47,6
- Met
- 60,6
- Leu
- 74,4
- Trp
- 85,3
- Ala
- 85,6
- Ile
- 87,6
- Arg
- 94,1
- Gly
- 100
- Ser
- 100
- Pro
- 100
- Asn
- 100
- Thr
- 100
- Gln
- 100
13
Claims (1)
-
imagen1 imagen2 imagen3
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| Application Number | Priority Date | Filing Date | Title |
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| EP09177095 | 2009-11-25 | ||
| EP09177095 | 2009-11-25 | ||
| US26751609P | 2009-12-08 | 2009-12-08 | |
| US267516P | 2009-12-08 | ||
| PCT/EP2010/068178 WO2011064282A1 (en) | 2009-11-25 | 2010-11-25 | Method for making polypeptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2550761T3 true ES2550761T3 (es) | 2015-11-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES10782288.4T Active ES2550761T3 (es) | 2009-11-25 | 2010-11-25 | Método para producción de polipéptidos |
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| Country | Link |
|---|---|
| US (1) | US8815541B2 (es) |
| EP (1) | EP2504355B1 (es) |
| JP (1) | JP5850848B2 (es) |
| CN (1) | CN102639559B (es) |
| ES (1) | ES2550761T3 (es) |
| WO (1) | WO2011064282A1 (es) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6121992B2 (ja) | 2011-06-10 | 2017-04-26 | ノヴォ ノルディスク アー／エス | ポリペプチド |
| JP6738326B2 (ja) * | 2014-06-06 | 2020-08-12 | ザ スクリプス リサーチ インスティテュート | フッ化硫黄（ｖｉ）化合物およびそれの製造方法 |
| BR112017019378A2 (pt) | 2015-03-18 | 2019-02-19 | Zealand Pharma A/S | análogos de amilina |
| EP3699269A1 (en) * | 2015-09-22 | 2020-08-26 | F. Hoffmann-La Roche AG | Expression of fc-containing proteins |
| CN105368864B (zh) * | 2015-12-24 | 2020-02-21 | 中国科学院海洋研究所 | 可溶性牙鲆npy重组蛋白获得方法 |
| US10071140B2 (en) | 2016-09-09 | 2018-09-11 | Zealand Pharma A/S | Amylin analogues |
| CN107400635A (zh) * | 2017-04-27 | 2017-11-28 | 广州弘宝元生物科技有限公司 | 导入α‑MSH多肽的粘红酵母重组菌株及其制备方法和应用 |
| SE2051317A1 (en) * | 2020-11-11 | 2022-05-12 | Nielsen Dina Petranovic | A genetically modified yeast cell |
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| US4599311A (en) | 1982-08-13 | 1986-07-08 | Kawasaki Glenn H | Glycolytic promotersfor regulated protein expression: protease inhibitor |
| US5242798A (en) * | 1983-07-21 | 1993-09-07 | Scripps Clinic And Research Foundation | Synthetic polypeptides corresponding to portions of proteinoids translated from brain-specific mRNAs, receptors, methods and diagnostics using the same |
| US4870008A (en) | 1983-08-12 | 1989-09-26 | Chiron Corporation | Secretory expression in eukaryotes |
| AU600885B2 (en) | 1984-05-25 | 1990-08-30 | Zymogenetics Inc. | Stable DNA constructs |
| US4931373A (en) | 1984-05-25 | 1990-06-05 | Zymogenetics, Inc. | Stable DNA constructs for expression of α-1 antitrypsin |
| US4766073A (en) | 1985-02-25 | 1988-08-23 | Zymogenetics Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
| US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
| US4882279A (en) | 1985-10-25 | 1989-11-21 | Phillips Petroleum Company | Site selective genomic modification of yeast of the genus pichia |
| US5367052A (en) | 1987-04-27 | 1994-11-22 | Amylin Pharmaceuticals, Inc. | Amylin peptides |
| GB8709871D0 (en) * | 1987-04-27 | 1987-06-03 | Turner R C | Peptides |
| US4929553A (en) | 1987-05-29 | 1990-05-29 | Canadian Patents & Development Ltd. | Protease for specific processing of secreted proteins |
| AU614121B2 (en) | 1988-05-04 | 1991-08-22 | Novartis Ag | Improvements in the production of polypeptides |
| GB8907110D0 (en) | 1988-05-04 | 1989-05-10 | Ciba Geigy Ag | Improvements in the production of polypeptides |
| US5013305A (en) | 1988-06-29 | 1991-05-07 | Opie Eric A | Needle safety system and method |
| US5037743A (en) | 1988-08-05 | 1991-08-06 | Zymogenetics, Inc. | BAR1 secretion signal |
| DK105489D0 (da) | 1989-03-03 | 1989-03-03 | Novo Nordisk As | Polypeptid |
| DK300090D0 (da) | 1990-12-19 | 1990-12-19 | Novo Nordisk As | Fremgangsmaade til fremstilling af leadersekvenser |
| GB9404270D0 (en) | 1994-03-05 | 1994-04-20 | Delta Biotechnology Ltd | Yeast strains and modified albumins |
| US5639642A (en) | 1994-06-16 | 1997-06-17 | Novo Nordisk A/S | Synthetic leader peptide sequences |
| IL114160A (en) | 1994-06-17 | 2006-12-31 | Novo Nordisk As | Dna constructs encoding heterologous proteins and processes for the heterologous protein production in yeast |
| CA2258307C (en) * | 1996-07-05 | 2008-02-12 | Novo Nordisk A/S | Method of producing heterologous short chain polypeptides by yeast with impaired yap3 protease |
| US6110703A (en) | 1996-07-05 | 2000-08-29 | Novo Nordisk A/S | Method for the production of polypeptides |
| AU6419099A (en) * | 1998-10-07 | 2000-04-26 | Children's Medical Center Corporation | Disruption of the kex1 gene in (pichia) and methods of full length protein expression |
| EP1534835B1 (en) * | 2002-06-29 | 2010-09-08 | Korea Research Institute of Bioscience and Biotechnology | Hansenula polymorpha yapsin deficient mutant strain and process for the preparation of recombinant proteins using the same |
| EP1546342A2 (en) * | 2002-09-20 | 2005-06-29 | Cym1P A/S | Methods for increasing the production of a recombinant polypeptide from a host cell |
| DK2038423T3 (da) * | 2006-06-21 | 2013-04-02 | Biocon Ltd | Fremgangsmåde til fremstilling af biologisk aktive polypeptider med insulinotropisk aktivitet |
-
2010
- 2010-11-25 ES ES10782288.4T patent/ES2550761T3/es active Active
- 2010-11-25 JP JP2012540424A patent/JP5850848B2/ja not_active Expired - Fee Related
- 2010-11-25 US US13/510,363 patent/US8815541B2/en active Active
- 2010-11-25 WO PCT/EP2010/068178 patent/WO2011064282A1/en not_active Ceased
- 2010-11-25 EP EP10782288.4A patent/EP2504355B1/en not_active Not-in-force
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Also Published As
| Publication number | Publication date |
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| EP2504355B1 (en) | 2015-07-22 |
| WO2011064282A1 (en) | 2011-06-03 |
| US8815541B2 (en) | 2014-08-26 |
| US20120282653A1 (en) | 2012-11-08 |
| CN102639559A (zh) | 2012-08-15 |
| JP2013511967A (ja) | 2013-04-11 |
| JP5850848B2 (ja) | 2016-02-03 |
| EP2504355A1 (en) | 2012-10-03 |
| CN102639559B (zh) | 2015-04-29 |
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