DK2147104T3 - Anvendelse af en asparaginprotease- (nsp24) signalsekvens til heterologt proteinekspression - Google Patents
Anvendelse af en asparaginprotease- (nsp24) signalsekvens til heterologt proteinekspression Download PDFInfo
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- DK2147104T3 DK2147104T3 DK08754611.5T DK08754611T DK2147104T3 DK 2147104 T3 DK2147104 T3 DK 2147104T3 DK 08754611 T DK08754611 T DK 08754611T DK 2147104 T3 DK2147104 T3 DK 2147104T3
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- host cell
- protein
- signal peptide
- heterologous protein
- sequence
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Classifications
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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
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
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- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
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- Biomedical Technology (AREA)
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- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Claims (16)
- ANVENDELSE AF EN ASPARAGINPROTEASE- (NSP24) SIGNALSEKVENS TIL HETEROLOGT PROTEINEKSPRESSION1. Fusionsprotein omfattende et signalpeptid bestående af aminosyreme MQTFGAFLVSFLAASGLAAA ifølge SEQ ID NO: 4, eller en variant deraf, der adskiller sig i sekvens med mellem 1 og 5 aminosyresubstitutioner, deletioner eller insertioner, og som er operativt forbundet med et heterologt protein, der ikke sekreteres naturligt af signalpeptidet, således at signalpeptidet bindes til en N-terminal ende af det heterologe protein, hvor varianten bevarer signalpeptidaktivitet ved at være i stand til at sekretere det heterologe protein fra en værtscelle.
- 2. Fusionsprotein ifølge krav 1, hvor det heterologe protein er udvalgt fra: en phytase, en glucoamylase, en alphaamylase, et granulært stivelseshydrolyserende enzym, en cellulase, en lipase, en xylanase, en cutinase, en hemicellulase, en protease, en oxidase eller en laccase.
- 3. Fusionsprotein ifølge krav 2, hvor det heterologe protein er en phytase med mindst 90 % sekvensidentitet med SEQ ID NO: 5.
- 4. Fusionsprotein ifølge et hvilket som helst af kravene 1 til 3, hvor signalpeptidet er en trunkeret variant af signalpeptidet ifølge SEQ ID NO: 4 med mindst 16 aminosyrer.
- 5. Isoleret polynukleotid omfattende et polynukleotid, der koder for fusionsproteinet ifølge et hvilket som helst af kravene 1 til 4.
- 6. Ekspressionsvektor omfattende et polynukleotid, der koder for fusionsproteinet ifølge et hvilket som helst af kravene 1 til 4, og eventuelt endvidere omfatter en promoter.
- 7. Værtscelle omfattende ekspressionsvektoren ifølge krav 6, hvor værtscellen er en svampeværtscelle.
- 8. Værtscelle ifølge krav 7, hvor værtscellen er en filamentøs svampecelle.
- 9. Værtscelle ifølge krav 7 eller krav 8, hvor værtscellen er en filamentøs svampecelle, der er en Aspergillus spp., en Fusarium spp. eller Trichoderma spp.
- 10. Værtscelle ifølge krav 9, hvor Aspergillus spp. er A. niger, A. oryzae, A. nidulans, eller A. awamori eller Trichoderma spp., der er T. reesei.
- 11. Værtscelle ifølge et hvilket som helst af kravene 7 til 10, hvor ekspressionsvektoren er stabilt integreret i værtscellens genom.
- 12. Fremgangsmåde til fremstilling af et heterologt protein i en svampeværtscelle, hvilken fremgangsmåde omfatter: tilvejebringelse af ekspressionsvektoren ifølge krav 6; transformering af værtscellen med ekspressionsvektoren; og dyrkning af værtscellen på betingelser, således at det heterologe protein udtrykkes og sekreteres fra værtscellen.
- 13. Fremgangsmåde ifølge krav 12, hvor svampeværtscellen er en filamentøs svampeværtscelle.
- 14. Fremgangsmåde ifølge krav 13, hvor den filamentøse svampeværtscelle er en Trichoderma- eller Aspergillus-ceUe.
- 15. Fremgangsmåde til fremstilling af et heterologt protein i en svampeværtscelle, hvilken fremgangsmåde omfatter introduktion i en værtscelle af et polynukleotid, der koder for et signalpeptid bestående af aminosyreme MQTFGAFLVSFLAASGLAAA (SEQ ID NO: 4), eller en variant deraf, der adskiller sig i sekvens med mellem 1 og 5 aminosyresubstitutioner, deletioner eller insertioner, der bevarer signalpeptidaktivitet ved at være i stand til at sekretere det heterologe protein fra en værtscelle, operativt forbundet med et heterologt protein, der ikke naturligt sekreteres, ved anvendelse af signalpeptidet, således at signalpeptidet bindes til en N-terminal ende af det heterologe protein, for at tilvejebringe en værtscelle ifølge et hvilket som helst af kravene 7 til 11, dyrkning af værtscellen under egnede dyrkningsbetingelser for ekspression og fremstilling af det heterologe protein, og fremstilling af det heterologe protein.
- 16. Fremgangsmåde ifølge krav 15, hvilken fremgangsmåde endvidere omfatter genvinding af det fremstillede heterologe protein.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US93107207P | 2007-05-21 | 2007-05-21 | |
| US98443007P | 2007-11-01 | 2007-11-01 | |
| PCT/US2008/001646 WO2008097619A2 (en) | 2007-02-07 | 2008-02-06 | Variant buttiauxella sp. phytases having altered properties |
| PCT/US2008/006498 WO2008150376A1 (en) | 2007-05-21 | 2008-05-21 | Use of an aspartic protease (nsp24) signal sequence for heterologous protein expression |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2147104T3 true DK2147104T3 (da) | 2015-12-14 |
Family
ID=39768616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK08754611.5T DK2147104T3 (da) | 2007-05-21 | 2008-05-21 | Anvendelse af en asparaginprotease- (nsp24) signalsekvens til heterologt proteinekspression |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US8093016B2 (da) |
| EP (1) | EP2147104B1 (da) |
| DK (1) | DK2147104T3 (da) |
| WO (1) | WO2008150376A1 (da) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006073839A2 (en) * | 2004-12-30 | 2006-07-13 | Genencor International, Inc. | Acid fungal proteases |
| GB0908770D0 (en) * | 2009-04-24 | 2009-07-01 | Danisco | Method |
| EP2446027A1 (en) * | 2009-06-25 | 2012-05-02 | Danisco A/S | Protein |
| WO2013007821A1 (en) * | 2011-07-14 | 2013-01-17 | Dsm Ip Assets B.V. | Screening method |
| WO2013181647A2 (en) | 2012-06-01 | 2013-12-05 | Danisco Us Inc. | Compositions and methods of producing isoprene and/or industrrial bio-products using anaerobic microorganisms |
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| WO2008150376A1 (en) | 2008-12-11 |
| EP2147104B1 (en) | 2015-09-09 |
| US20110027830A1 (en) | 2011-02-03 |
| EP2147104A1 (en) | 2010-01-27 |
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