DK2421973T3 - Proteaser med modificerede pro-områder - Google Patents
Proteaser med modificerede pro-områder Download PDFInfo
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- DK2421973T3 DK2421973T3 DK10714540.1T DK10714540T DK2421973T3 DK 2421973 T3 DK2421973 T3 DK 2421973T3 DK 10714540 T DK10714540 T DK 10714540T DK 2421973 T3 DK2421973 T3 DK 2421973T3
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- DK
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- Prior art keywords
- protease
- seq
- mature
- polynucleotide
- bacillus
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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Claims (12)
- PROTEASER MED MODIFICEREDE PRO-OMRÅDER1. Anvendelse af et modificeret polynukleotid, der koder for en modificeret fuldlængde-protease, hvilket modificeret polynukleotid omfatter: et første polynukleotid, der koder for et signalpeptid, hvor det første polynukleotid er operativt bundet til et andet polynukleotid, der koder for pro-området ifølge SEQ ID NO: 7, hvor pro-området omfatter en kombination af substitutioner af mindst to aminosyrer ved positioner, der er udvalgt fra positionerne 6, 30 og 32 af pro-området, hvor kombinationen af substitutioner er udvalgt fra E6R-A32K, E6N-A32K, E6D-A32K, E6I-A32K, E6K-A32K, E6M-A32K, E6P-A32K, E6T-A32K, E6N-A32K, E6A-E30G, E6R-E30G, E6C-E30G, E6Q-E30G, E6G-E30G, E6H-E30G, E6K-E30G, E6S-E30G, E6W-E30G, E30G-A32R, E30G-A32Q, E30G-A32E, E30G-A32G, E30G-A32H, E30G-A32I, E30G-A32S, E30G-A32T, E30G-A32W, E30G-A32V, E6G-E30G-A32E, E6G-E30G-A32S, E6G-E30G-A32T, E6G-E30G-A32W, E6A-E30G, E6R-E30G, E6N-E30G, E6D-E30G, E6C-E30G, E6Q-E30G, E6G-E30G, E6H-E30G, E6K-E30G, E6M-E30G, E6F-E30G, E6P-E30G, E6S-E30G, E6T-E30G, E6W-E30G, E6V-E30G, E6Y-E30G, E6A-E30S, E6G-E30S, E6L-E30S, E6K-E30S, E6F-E30S, E6P-E30S, E6Y-E30S, E6V-E30S, E30S-A32R, E30S-A32N, E30S-A32D, E30S-A32C, E30S-A32Q, E30S-A32E, E30S-A32G, E30S-A32H, E30S-A32L, E30S-A32M, E30S-A32F, E30S-A32P, E30S-A32S, E30S-A32T, E30S-A32W, E30S-A32Y og E30S-A32V, og hvor positionerne er nummereret i overensstemmelse med aminosyresekvensen for pro-polypeptidet ifølge SEQ ID NO: 7; hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af en protease, der er mindst ca. 60 % identisk med den modne protease ifølge SEQ ID NO: 9, for at tilvejebringe en forbedret produktion af en moden protease, der er oparbejdet fra den modificerede, fuldlængde-protease, sammenlignet med produktionsniveauet af den samme modne protease, når den er oparbejdet fra fuldlængde-proteasen uden substitutionerne i pro-området ifølge SEQ ID NO: 7.
- 2. Anvendelse ifølge krav 1, hvor den modne protease er en alkalisk vildtype serinprotease eller serinproteasevariant, der er afledt af Bacillus clausii eller Bacillus lentus.
- 3. Anvendelse ifølge krav 1 eller krav 2, hvor den modne protease har en aminosyresekvens, der er udvalgt fra SEQ ID NO: 9, 11, 13, 15, 17, 19 og 21.
- 4. Anvendelse ifølge et hvilket som helst af kravene 1-3, hvor signalpeptidet har en aminosyresekvens, der er udvalgt fra SEQ ID NO: 3 og 5.
- 5. Anvendelse ifølge et hvilket som helst af kravene 1-4, hvor substitutionerne forbedrer produktionen af den modne protease ved hjælp af en Bacillus sp. værtscelle.
- 6. Anvendelse ifølge krav 5, hvor Bacillus sp. værtscellen er en Bacillus subtilis-væriscelle.
- 7. Fremgangsmåde til forbedring af produktion af en moden protease i en Bacillus sp. værtscelle, hvilken fremgangsmåde omfatter: a) tilvejebringelse af en ekspressionsvektor, der omfatter det modificerede polynukleotid som defineret i et hvilket som helst af kravene 1 til 4; b) transformering af en Bacillus sp. værtscelle med ekspressionsvektoren; og c) dyrkning af værtscellen under egnede forhold, således at den modne protease produceres af værtscellen; hvor produktion af den modne protease, der er oparbejdet fra den modificerede, fuldlængde -protease, er forbedret sammenlignet med produktionsniveauet af den samme modne protease, når den er oparbejdet fra fuldlængde-proteasen uden substitutionerne i pro-området ifølge SEQ ID NO: 7; eventuelt hvor Bacillus sp. værtscellen er en Bacillus subtilis-værtscelle.
- 8. Fremgangsmåde ifølge krav 7, hvor ekspressionsvektoren endvidere omfatter en AprE-promoter.
- 9. Fremgangsmåde ifølge krav 7 eller 8, hvor den modne protease er en alkalisk vildtype -Bacillus clausii- eller en Bacillus lentus-serin protease, variant eller homolog deraf.
- 10. Fremgangsmåde ifølge et hvilket som helst af kravene 7 til 9, hvor det første polynukleotid koder for signalpeptidet ifølge SEQ ID NO: 3, hvor: a) det andet polynukleotid koder for pro-området omfatter en kombination af substitutioner, der er udvalgt fra: E6R-A32K, E6N-A32K, E6D-A32K, E6I-A32K, E6K-A32K, E6M-A32K, E6P-A32K, E6T-A32K og E6N-A32K, og hvor det tredje polynukleotid koder for en moden protease, der er udvalgt fra SEQ ID NO: 9; eller b) det andet polynukleotid, der koder for pro-området, omfatter en kombination af substitutioner, der er udvalgt fra E6A-E30G, E6R-E30G, E6C-E30G, E6Q-E30G, 1:6(,-5 E30G, E6H-E30G, E6K-E30G, E6S-E30G, E6W-E30G, E30G-A32R, E30G-A32Q, E30G-A32E, E30G-A32G, E30G-A32H, E30G-A32I, E30G-A32S, E30G-A32T, E30G-A32W, E30G-A32V, E6G-E30G-A32E, E6G-E30G-A32S, E6G-E30G-A32T og E6G E30G-A32W, og hvor det tredje polynukleotid koder for en moden protease, der er udvalgt fra SEQ ID NO: 17; eller c) det andet polynukleotid, der koder for pro-området, omfatter en kombination af substitutioner, der er udvalgt fra E6A-E30G, E6R-E30G, E6N-E30G, E6D-E30G, E6C-E30G, E6Q-E30G, E6G-E30G, E6H-E30G, E6K-E30G, E6M-E30G, E6F-E30G, E6P-E30G, E6S-E30G, E6T-E30G, E6W-E30G, E6V-E30G og E6Y-E30G, og hvor det tredje polynukleotid koder for en moden protease, der er udvalgt fra SEQ ID NO: 19; eller d) det andet polynukleotid, der koder for pro-området, omfatter en kombination af substitutioner, der er udvalgt fra E6A-E30S, E6G-E30S, E6L-E30S, E6K-E30S, E6F-E30S, E6P-E30S, E6Y-E30S, E6V-E30S, E30S-A32R, E30S-A32N, E30S-A32D, E30S-A32C, E30S-A32Q, E30S-A32E, E30S-A32G, E30S-A32H, E30S-A32L, E30S-A32M, E30S-A32F, E30S-A32P, E30S-A32S, E30S-A32T, E30S-A32W, E30S-A32Y og E30S-A32V, og hvor det tredje polynukleotid koder for en moden protease, der er udvalgt fra SEQ ID NO: 21.
- 11. Anvendelse af et polynukleotid, der koder for en modificeret fuldlængde-protease, hvor det isolerede, modificerede polynukleotid omfatter et første polynukleotid, der koder for signalpeptidet ifølge SEQ ID NO: 3, hvor det første polynukleotid er operativt bundet til et andet polynukleotid, der koder for pro-området ifølge SEQ ID NO: 7, hvor: a) pro-området omfatter en substitution af en aminosyre, der er udvalgt fra E6A, E6C, E6Y, E30A, E30L, E30P, E30T, E30Y, E30V, A32C, A32E, A32G, A32K og A32T, hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af proteasen ifølge SEQ ID NO: 17; eller b) pro-området omfatter en substitution af en aminosyre, der er udvalgt fra E6A, E6C, E6F, E6P, E6T, E6W, E6V, A32K og A32T, hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af proteasen ifølge SEQ ID NO: 9; eller c) pro-området omfatter en substitution af en aminosyre, der er udvalgt fra E6A, E30A, E30L, E30T og E30V, hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af proteasen ifølge SEQ ID NO: 19; eller d) pro-området omfatter en substitution af en aminosyre, der er udvalgt fra E6A, 5 E6L, E6F, E6T, E6V, E30I, E30L, E30P, E30T, E30Y, E30V, A32Q, A32S og A32T, hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af proteasen ifølge SEQ ID NO: 11; eller e) pro-området omfatter the aminosyre substitution E30A, hvor det andet polynukleotid er operativt bundet til et tredje polynukleotid, der koder for det modne område af proteasen ifølge SEQ ID NO: 21, for at tilvejebringe forbedret produktion af en moden protease, der er oparbejdet fra den modificerede, fuldlængde-protease, sammenlignet med produktionsniveauet af den samme modne protease, når den er oparbejdet fra fuldlængde-proteasen uden substitutionerne i pro-området ifølge SEQ ID NO: 7.
- 12. Fremgangsmåde til forbedring af produktion af en moden protease i en Bacillus subtilis-værtsceWe, hvilken fremgangsmåde omfatter a) tilvejebringelse af en ekspressionsvektor, der omfatter polynukleotidet som defineret i krav 11; b) transformering af Bacillus sz/Az/V/b-værtscel len med ekspressionsvektoren; og c) dyrkning af værtscellen under egnede forhold, således at den modne protease produceres af værtscellen; hvor produktion af den modne protease, der er oparbejdet fra den modificerede, fuldlængde -protease er forbedret, sammenlignet med produktionsniveauet af den samme modne protease, når den er oparbejdet fra fuldlængde-proteasen uden substitutionerne i pro-området ifølge SEQ ID NO: 7.
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| EP2129779B2 (en) * | 2007-03-12 | 2018-12-26 | Danisco US Inc. | Modified proteases |
| AR076312A1 (es) * | 2009-04-24 | 2011-06-01 | Danisco Us Inc | Proteasas con regiones pro modificadas |
| CN102575242B (zh) * | 2009-07-31 | 2015-03-25 | 丹尼斯科美国公司 | 具有经修饰的前原区域的蛋白酶 |
| GB0915572D0 (en) | 2009-09-07 | 2009-10-07 | Reckitt Benckiser Nv | Detergent composition |
| PL2558573T3 (pl) * | 2010-04-15 | 2017-08-31 | Danisco Us Inc. | Kompozycje i sposoby obejmujące warianty proteazy |
| US20120067373A1 (en) * | 2010-04-15 | 2012-03-22 | Philip Frank Souter | Automatic Dishwashing Detergent Composition |
| WO2013086219A1 (en) | 2011-12-09 | 2013-06-13 | Danisco Us Inc. | Ribosomal promotors from b. subtilis for protein production in microorganisms |
| GB201212932D0 (en) * | 2012-07-20 | 2012-09-05 | Dupont Nutrition Biosci Aps | Method |
| GB201212934D0 (en) * | 2012-07-20 | 2012-09-05 | Dupont Nutrition Biosci Aps | Method |
| US10030221B2 (en) | 2012-09-20 | 2018-07-24 | Danisco Us Inc. | Microtiter plates for controlled release of culture components to cell cultures |
| US10550381B2 (en) | 2014-07-04 | 2020-02-04 | Novozymes A/S | Variant proteases and amylases having enhanced storage stability |
| EP3739029A1 (en) | 2014-07-04 | 2020-11-18 | Novozymes A/S | Subtilase variants and polynucleotides encoding same |
| CN107075491B (zh) * | 2014-10-28 | 2021-07-06 | 谷万达公司 | 用于稳定反式剪接的内含肽修饰的蛋白酶的方法和组合物 |
| US20180030456A1 (en) | 2015-02-19 | 2018-02-01 | Danisco Us Inc. | Enhanced protein expression |
| WO2016205710A1 (en) * | 2015-06-17 | 2016-12-22 | Danisco Us Inc. | Proteases with modified propeptide regions |
| WO2017089093A1 (en) | 2015-11-25 | 2017-06-01 | Unilever N.V. | A liquid detergent composition |
| CN109071615A (zh) | 2016-03-04 | 2018-12-21 | 丹尼斯科美国公司 | 用于在微生物中产生蛋白质的工程化核糖体启动子 |
| CN110709513B (zh) * | 2017-04-11 | 2023-10-03 | 豪夫迈·罗氏有限公司 | 具有热稳定性增加的突变型逆转录酶以及涉及其的产物、方法和用途 |
| US20200354708A1 (en) | 2017-11-29 | 2020-11-12 | Danisco Us Inc. | Subtilisin variants having improved stability |
| US12509672B2 (en) | 2018-11-28 | 2025-12-30 | Danisco Us Inc. | Subtilisin variants having improved stability |
| EP3935160A1 (en) * | 2019-03-08 | 2022-01-12 | Novozymes A/S | Means and methods for improving protease expression |
| EP4504756A1 (en) * | 2022-04-01 | 2025-02-12 | Danisco US Inc. | Pro-region mutations enhancing protein production in gram-positive bacterial cells |
| EP4525615A2 (en) | 2022-05-14 | 2025-03-26 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
| CN116891844A (zh) * | 2023-06-07 | 2023-10-17 | 湖北佶凯星生物科技有限公司 | 一种碱性蛋白酶高效融合表达策略 |
| WO2025034713A2 (en) | 2023-08-09 | 2025-02-13 | Danisco Us Inc. | Compositions and methods for enhanced protein production in gram‑positive bacterial cells |
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| US4302544A (en) | 1979-10-15 | 1981-11-24 | University Of Rochester | Asporogenous mutant of B. subtilis for use as host component of HV1 system |
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| CA2324422A1 (en) * | 1998-03-26 | 1999-09-30 | The Procter & Gamble Company | Serine protease variants having amino acid deletions and substitutions |
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| US8535927B1 (en) * | 2003-11-19 | 2013-09-17 | Danisco Us Inc. | Micrococcineae serine protease polypeptides and compositions thereof |
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| AR076312A1 (es) * | 2009-04-24 | 2011-06-01 | Danisco Us Inc | Proteasas con regiones pro modificadas |
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| JP2012524542A (ja) | 2012-10-18 |
| US20160040147A1 (en) | 2016-02-11 |
| US9115351B2 (en) | 2015-08-25 |
| US8530218B2 (en) | 2013-09-10 |
| EP2421973B1 (en) | 2018-05-23 |
| WO2010123754A1 (en) | 2010-10-28 |
| AR076312A1 (es) | 2011-06-01 |
| CN102414321A (zh) | 2012-04-11 |
| CA2759695A1 (en) | 2010-10-28 |
| CA2759695C (en) | 2018-05-01 |
| US20140045268A1 (en) | 2014-02-13 |
| US20110045571A1 (en) | 2011-02-24 |
| US9593320B2 (en) | 2017-03-14 |
| EP2421973A1 (en) | 2012-02-29 |
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