HRP20110084T2 - Promotori mutantne aox1 - Google Patents
Promotori mutantne aox1 Download PDFInfo
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- HRP20110084T2 HRP20110084T2 HR20110084T HRP20110084T HRP20110084T2 HR P20110084 T2 HRP20110084 T2 HR P20110084T2 HR 20110084 T HR20110084 T HR 20110084T HR P20110084 T HRP20110084 T HR P20110084T HR P20110084 T2 HRP20110084 T2 HR P20110084T2
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- 239000002773 nucleotide Substances 0.000 claims abstract 49
- 125000003729 nucleotide group Chemical group 0.000 claims abstract 49
- 101710194180 Alcohol oxidase 1 Proteins 0.000 claims abstract 15
- 241000235058 Komagataella pastoris Species 0.000 claims abstract 9
- 102000040945 Transcription factor Human genes 0.000 claims abstract 8
- 108091023040 Transcription factor Proteins 0.000 claims abstract 8
- 230000035772 mutation Effects 0.000 claims abstract 6
- 210000004027 cell Anatomy 0.000 claims 25
- 150000007523 nucleic acids Chemical class 0.000 claims 22
- 102000039446 nucleic acids Human genes 0.000 claims 21
- 108020004707 nucleic acids Proteins 0.000 claims 21
- 241000235648 Pichia Species 0.000 claims 11
- 108090000623 proteins and genes Proteins 0.000 claims 10
- 238000000034 method Methods 0.000 claims 8
- 102000004169 proteins and genes Human genes 0.000 claims 8
- 210000005253 yeast cell Anatomy 0.000 claims 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 6
- 235000018102 proteins Nutrition 0.000 claims 6
- 239000003550 marker Substances 0.000 claims 5
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 2
- 101100523604 Mus musculus Rassf5 gene Proteins 0.000 claims 2
- 108010084455 Zeocin Proteins 0.000 claims 2
- 101150067149 abaA gene Proteins 0.000 claims 2
- 229910052799 carbon Inorganic materials 0.000 claims 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 claims 2
- 238000002955 isolation Methods 0.000 claims 2
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 claims 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims 1
- 229930195725 Mannitol Natural products 0.000 claims 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims 1
- -1 Stre Proteins 0.000 claims 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims 1
- 235000004279 alanine Nutrition 0.000 claims 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims 1
- 239000003242 anti bacterial agent Substances 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 101150038738 ble gene Proteins 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 claims 1
- 230000037430 deletion Effects 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 238000004520 electroporation Methods 0.000 claims 1
- 210000003527 eukaryotic cell Anatomy 0.000 claims 1
- 230000004927 fusion Effects 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000037431 insertion Effects 0.000 claims 1
- 238000003780 insertion Methods 0.000 claims 1
- 239000008101 lactose Substances 0.000 claims 1
- 239000000594 mannitol Substances 0.000 claims 1
- 235000010355 mannitol Nutrition 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 claims 1
- 239000000600 sorbitol Substances 0.000 claims 1
- 235000010356 sorbitol Nutrition 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 230000009466 transformation Effects 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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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
- C12N15/79—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
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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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
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03013—Alcohol oxidase (1.1.3.13)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
Promotor alkoholne oksidaze 1 mutantne Pichia pastoris (AOX1), naznačen time, da je to promotor divljeg tipa Pichia pastoris AOX1 (SEQ ID No.1) koji sadrži najmanje jednu mutaciju unutar nukleotida 170 do 235 (-784 do -719) od SEQ ID No.1 za visoku ekspresiju pod uvjetima induciranog metanola. Patent sadrži još 18 patentnih zahtjeva.
Claims (19)
1. Promotor alkoholne oksidaze 1 mutantne Pichia pastor is (AOX1), naznačen time, da je to promotor divljeg tipa Pichia pastor is AOX1 (SEQ ID No.l) koji sadrži najmanje jednu mutaciju unutar nukleotida 170 do 235 (-784 do -719) od SEQ ID No.l za visoku ekspresiju pod uvjetima induciranog metanola.
2. Promotor prema zahtjevu 1, naznačen time, da promotor sadrži nadalje mutaciju nukleotida 694-723 (-260 do -231) i/ili nukleotida 729 do 763 (-225 do -191) od SEQ ID No.l i/ili od faktora transkripcije na mjestu vezivanja (TFBS) i/ili najmanje jednu mutaciju odabranu iz skupine koja sadrži nukleotide 170 do 191 (-784 do -763), nukleotide 192 do 213 (-762 do -741), nukleotide 192 do 210 (-762 do -744), nukleotide 207 do 209 (-747 do -745), nukleotide 214 do 235 (-740 do -719), nukleotide 304 do 350 (-650 do -604), nukleotide 364 do 393 (-590 do -561), nukleotide 434 do 508 (-520 do -446), nukleotide 509 do 551 (-445 do -403), nukleotide 552 do 560 (-402 do -394), nukleotide 585 do 617 (-369 do -337), nukleotide 621 do 660 (-333 do -294), nukleotide 625 do 683 (-329 do -271), nukleotide 736 do 741 (-218 do -213), nukleotide 737 do 738 (-217 do -216), nukleotide 726 do 755 (-228 do -199), nukleotide 784 do 800 (-170 do -154) ili nukleotide 823 do 861 (-131 do -93).
3. Promotor prema zahtjevu 1 ili 2, naznačen time, da mutacija je izbacivanje, supstitucija, umetanje ili inverzija.
4. Promotor prema zahtjevu 2 ili 3, naznačen time, da je faktor transkripcije na mjestu spajanja (TFBS) odabran iz skupine koja sadrži Hapl, Hsf, Hap234, abaA, Stre, Rapl, Adrl, MatlMC, Gcrl i QA-1F, pri čemu faktor transkripcije na mjestu spajanja (TFBS) Hapl sadrži prednosno nekleotide 54 do 58 od SEQ ID No.l. Hsf nukleotide 142 do 149 i 517 do 524 od SEQ ID No.l, Hap234 nukleotide 196 do 200, 206 do 210 i 668 do 672 od SEQ ID No.l, abaA nukleotide 219 do 224 od SEQ ID No.l, Stre nukleotide 281 do 285 od SEQ ID No.l, Rapl nukleotide 335 do 339 od SEQ ID No.l, Adrl nukleotide 371 do 377 od SEQ ID No.l, MatlMC nukleotide 683 do 687 od SEQ ID No.l, Gcrl nukleotide 702 do 706 od SEQ ID No.l i QA-1F nukleotide 747 do 761 odSEQIDNo.l.
5. Molekula nukleinske kiseline koja sadrži najmanje jedan promotor alkoholne oksidaze 1 mutantne Pichiapastoris (AOX1) prema bilo kojem zahtjevu 1 do 4, i najmanje jednu nukleinsku kiselinu koja kodira protein (peptid) ili funkcionalnu nukleinsku kiselinu, naznačena time, da su spomenuti promotor i spomenuta nukleinska kiselina operativno povezani zajedno formirajući jednostruku ili višestruku kopiju kasete ekspresije.
6. Vektor, naznačen time, da sadrži promotor alkoholne oksidaze 1 mutantne Pichia pastoris (AOX1) prema bilo kojem zahtjevu 1 do 4, ili molekulu nukleinske kiseline prema zahtjevu 5.
7. Stanica, naznačena time, da sadrži najmanje jedan promotor alkoholne oksidaze 1 mutantne Pichia pastoris (AOX1) prema bilo kojem zahtjevu 1 do 4, najmanje jedan dio nukleinske kiseline prema zahtjevu 5 ili najmanje jedan vektor prema zahtjevu 6.
8. Stanica prema zahtjevu 7, naznačena time, da spomenuta stanica je eukariotička stanica, posebno stanica kvasca, prednosno metilotrofička stanica kvasca, najbolje odabrana iz skupine koja se sastoji od stanica Candida, Hansenula, Pichia i Toruplosis, posebno stanica Pichia pastoris.
9. Garnitura za ekspresiju odabranog proteina, naznačena time, da sadrži
(i) vektor prema zahtjevu 6, i
(ii) (ii) stanicu sposobnu za ekspresiju spomenutog proteina pod kontrolom promotora prema bilo kojem zahtjevu od 1 do 4.
10. Garnitura prema zahtjevu 9, naznačena time, da spomenuta stanica je stanica kvasca, najbolje metilotrofička stanica kvasca, prednosno odabrana iz skupine koja se sastoji od stanica Candida, Hansenula, Pichia i Toruplosis, posebno stanica Pichia pastor is.
11. Postupak ekspresije rekombinantnog proteina, peptida ili funkcionalne nukleinske kiseline u stanici, naznačen time, da sadrži sljedeće korake:
- dobavljanje molekule nukleinske kiseline prema zahtjevu 5 ili vektora prema zahtjevu 6 koji sadrži promotor AOX1 prema bilo kojem zahtjevu od 1 do 4 i nukleinsku kiselinu koja kodira za protein, peptid ili funkcionalnu nukleinsku kiselinu, pri čemu je spomenuti promotor operativno povezan na spomenutu nukleinsku kiselinu,
- transformiranje spomenute stanice sa spomenutim vektorom ili spomenutom molekulom nukleinske kiseline,
- uzgajanje transformirane stanice u pogodnom mediju za uzgoj,
- opcijski induciranje ekspresije spomenutog proteina, peptida ili funkcionalne nukleinske kiseline i
- izoliranje spomenutog izraženog proteina, peptida ili funkcionalne nukleinske kiseline.
12. Postupak prema zahtjevu 11,naznačen time, da spomenuta stanica je stanica kvasca, najbolje metilotrofička stanica kvasca, prednosno odabrana iz skupine koja se sastoji od stanica Candida, Hansenula, Pichia i Toruplosis, posebno stanica Pichia pastoris.
13. Uporaba molekule nukleinske kiseline prema zahtjevu 5, vektora prema zahtjevu 6 ili stanice prema bilo kojem zahtjevu 7 ili 8, naznačena time, da je za ekspresiju proteina, peptida ili funkcionalne nukleinske kiseline.
14. Postupak izolacije prvorazredne ekspresije klonova, naznačen time, da sadrži sljedeće korake:
a) uvođenje molekule nukleinske kiseline koja sadrži najmanje jednu mutaciju promotora alkoholne oksidaze 1 mutantne Pichia pastoris (AOX1) prema bilo kojem zahtjevu od 1 do 4 i najmanje jednu nukleinsku kiselinu koja kodira protein (peptid) ili funkcionalnu nukleinsku kiselinu i marker otpornosti gena, pri čemu su spomenuti promotor i spomenuta nukleinska kiselina operativno povezani zajedno formirajući jednostruku ili višestruku kopiju kasete ekspresije ili vektor koji sadrži molekulu nukleinske kiseline u stanici,
b) prenošenje stanice iz koraka (a) u medij koji sadrži prikladan selektivni marker, neograničavajući izvor ugljika i metanol za selektivni rast klonova prvorazredne ekspresije pod induciranim uvjetima,
c) inkubacija stanice iz koraka (b) na spomenutom mediju,
d) izolacija kolonije stanica dobivenih iz koraka (c),
e) otkrivanje klonova prvorazredne ekspresije određivanjem razmjera ekspresije spomenute stanice.
15. Postupak prema zahtjevu 14, naznačen time, da selektivni marker je antibiotik, prednosno Zeocin ili geneticin.
16. Postupak prema zahtjevu 14 ili 15, naznačen time, da selektivni marker je Zeocin i marker otpornosti gena je gen sh ble.
17. Postupak prema bilo kojem zahtjevu od 14 do 16, naznačen time, da stanica je stanica kvasca, prednosno metilotrofička stanica kvasca, najbolje odabrana iz skupine koja se sastoji od stanica Candida, Hansenula, Pichia i Toruplosis, posebno stanica Pichia pastoris.
18. Postupak prema bilo kojem zahtjevu od 11 do 17, naznačen time, da je neograničavajući izvor ugljika odabran iz skupine koja sadrži alanin, manitol, sorbitol, trehaloza, laktoza i njihova kombinacija.
19. Postupak prema bilo kojem zahtjevu od 11 do 18, naznačen time, da je molekula nukleinske kiseline ili vektor uveden u stanicu putem transformacije, najbolje elektroporacijom ili kemijskom transformacijom, ili putem protoplastičke fuzije, ili putem bombardiranja čestica.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0030405A AT501955B1 (de) | 2005-02-23 | 2005-02-23 | Mutierte aox1-promotoren |
| PCT/AT2006/000079 WO2006089329A2 (en) | 2005-02-23 | 2006-02-23 | Mutant aox 1 promoters |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HRP20110084T1 HRP20110084T1 (hr) | 2011-03-31 |
| HRP20110084T2 true HRP20110084T2 (hr) | 2011-10-31 |
Family
ID=36927772
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HR20110084T HRP20110084T2 (hr) | 2005-02-23 | 2011-02-03 | Promotori mutantne aox1 |
| HR20110254T HRP20110254T1 (hr) | 2005-02-23 | 2011-04-08 | Promotori mutantne aox1 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HR20110254T HRP20110254T1 (hr) | 2005-02-23 | 2011-04-08 | Promotori mutantne aox1 |
Country Status (21)
| Country | Link |
|---|---|
| US (2) | US9012175B2 (hr) |
| EP (2) | EP2199389B1 (hr) |
| JP (2) | JP5497986B2 (hr) |
| KR (2) | KR101352399B1 (hr) |
| CN (2) | CN101128581B (hr) |
| AT (3) | AT501955B1 (hr) |
| AU (2) | AU2006216094B2 (hr) |
| BR (1) | BRPI0606179B1 (hr) |
| CA (2) | CA2785161C (hr) |
| DE (2) | DE602006019789D1 (hr) |
| DK (2) | DK2199389T3 (hr) |
| ES (2) | ES2358086T3 (hr) |
| HK (2) | HK1113172A1 (hr) |
| HR (2) | HRP20110084T2 (hr) |
| IN (1) | IN2012DN06694A (hr) |
| NZ (2) | NZ560542A (hr) |
| PL (2) | PL1851312T3 (hr) |
| PT (1) | PT1851312E (hr) |
| SI (2) | SI2199389T1 (hr) |
| UA (2) | UA94399C2 (hr) |
| WO (1) | WO2006089329A2 (hr) |
Families Citing this family (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2271755B1 (en) * | 2008-03-27 | 2017-03-22 | E. I. du Pont de Nemours and Company | High expression zymomonas promoters |
| WO2010004042A2 (en) * | 2008-07-11 | 2010-01-14 | Novozymes A/S | Pichia pastoris das promoter variants |
| EP2258855A1 (en) | 2009-05-28 | 2010-12-08 | Universität für Bodenkultur Wien | Expression sequences |
| WO2012129036A2 (en) * | 2011-03-22 | 2012-09-27 | Merck Sharp & Dohme Corp. | Promoters for high level recombinant expression in fungal host cells |
| GB201113419D0 (en) * | 2011-08-04 | 2011-09-21 | Fujifilm Diosynth Biotechnologies Uk Ltd | Yeast vector |
| EP2565262A1 (en) | 2011-08-31 | 2013-03-06 | VTU Holding GmbH | Protein expression |
| KR101954389B1 (ko) * | 2011-10-07 | 2019-03-06 | 론자 리미티드 | 조절가능한 프로모터 |
| US9309523B2 (en) | 2012-12-05 | 2016-04-12 | Exxonmobil Research And Engineering Company | Nannochloropsis kozak consensus sequence |
| EP3594351B1 (en) | 2013-03-08 | 2021-09-29 | Keck Graduate Institute of Applied Life Sciences | Yeast promoters from pichia pastoris |
| CN105358706A (zh) * | 2013-03-08 | 2016-02-24 | 生物语法公司 | 用于蛋白质表达的酵母启动子 |
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| EP1431387B1 (en) * | 2002-12-20 | 2008-02-27 | Roche Diagnostics GmbH | Heat-labile desoxyribonculease I variants |
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