DK2993231T3 - Bakteriestamme til rekombinant proteinekspression med proteasefattig DEGP-tilbageholdende chaperoneaktivitet og deaktiverede Tsp- og ptr-gener - Google Patents

Bakteriestamme til rekombinant proteinekspression med proteasefattig DEGP-tilbageholdende chaperoneaktivitet og deaktiverede Tsp- og ptr-gener Download PDF

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DK2993231T3
DK2993231T3 DK15182886.0T DK15182886T DK2993231T3 DK 2993231 T3 DK2993231 T3 DK 2993231T3 DK 15182886 T DK15182886 T DK 15182886T DK 2993231 T3 DK2993231 T3 DK 2993231T3
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cell
gene
mutated
protein
ala
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DK15182886.0T
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Mark Ellis
David Paul Humphreys
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Ucb Biopharma Sprl
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Claims (23)

1. Rekombinant gramnegativ bakteriecelle omfattende: a. et muteret Tsp-gen, hvor det muterede Tsp-gen koder for et Tsp-protein med 50 % eller mindre af proteaseaktiviteten hos et ikke-muteret vildtype-Tsp eller er et de-aktiveret muteret Tsp-gen; hvor cellen er isogen med en celle af E. coli W3110 på nær det muterede Tsp-gen og eventuelt en polynukleotidsekvens kodende for et protein af interesse.
2. Celle ifølge krav 1, hvor det deaktiverede muterede Tsp-gen omfatter en mutation i genstartkodonen og/eller en eller flere stopkodoner placeret nedstrøms for genstartkodo-nen og opstrøms for genstopkodonen.
3. Celle ifølge kravene 1 eller 2, hvor cellen omfatter et deaktiveret muteret Tsp-gen.
4. Celle ifølge et hvilket som helst af kravene 1 til 3, hvor cellen er stamme MXE001 deponeret hos National Collection of Type Cultures, ΗΡΑ, Det forenede Kongerige, under deponeringsnummer NCTC13444.
5. Rekombinant gramnegativ bakteriecelle omfattende: a. et muteret Tsp-gen, hvor det muterede Tsp-gen koder for et Tsp-protein med 50 % eller mindre af proteaseaktiviteten hos et ikke-muteret vildtype-Tsp eller er et deaktiveret muteret Tsp-gen; og b. et muteret DegP-gen kodende for et DegP-protein med chaperoneaktivitet men ikke proteaseaktivitet, hvor cellen er isogen med en celle af E. coli W3110 på nær de muterede Tsp- og DegP-gener og eventuelt en polynukleotidsekvens kodende for et protein af interesse.
6. Celle ifølge krav 5, hvor cellen er stamme MXE005 deponeret hos National Collection of Type Cultures, ΗΡΑ, Det forenede Kongerige, under deponeringsnummer NCTC13448.
7. Rekombinant gramnegativ bakteriecelle omfattende: a. et muteret Tsp-gen, hvor det muterede Tsp-gen koder for et Tsp-protein med 50 % eller mindre af proteaseaktiviteten hos et ikke-muteret vildtype-Tsp eller er et deaktiveret muteret Tsp-gen; og b. et deaktiveret muteret ptr-gen, hvor cellen er isogen med en celle af E. coli W3110 på nær de muterede Tsp- og ptr-gener og eventuelt en polynukleotidsekvens kodende for et protein af interesse.
8. Celle ifølge krav 7, hvor cellen er stamme MXE004 deponeret hos National Collection of Type Cultures, ΗΡΑ, Det forenede Kongerige, under deponeringsnummer NCTC13447.
9. Celle ifølge et hvilket som helst af de foregående krav, hvor det muterede DegP-gen, det muterede ptr-gen og/eller det muterede Tsp-gen er muteret til at omfatte et eller flere restriktionsmarkørsteder.
10. Celle ifølge krav 9, hvor det deaktiverede muterede ptr-gen og/eller det deaktiverede muterede Tsp-gen er muterede til at omfatte et restriktionsmarkørsted omfattende en stopkodon i rammen.
11. Celle ifølge krav 9, hvor restriktionsmarkørstedet er et Ase /-restriktionssted.
12. Celle ifølge et hvilket som helst af kravene 9 til 11, hvor det deaktiverede muterede ptr-gen og/eller det deaktiverede muterede Tsp-gen omfatter et restriktionsmarkørsted dannet af en missense-mutation i genstartkodonen og eventuelt en eller flere punktmutationer.
13. Celle ifølge krav 12, hvor restriktionsmarkørstedet er et EcoR J-markørsted.
14. Celle ifølge et hvilket som helst af kravene 7 eller 9 til 13, hvor det deaktiverede muterede ptr-gen omfatter SEQ ID NO:6.
15. Celle ifølge et hvilket som helst af de foregående krav, hvor det deaktiverede muterede Tsp-gen omfatter SEQ ID NO:3.
16. Celle ifølge et hvilket som helst af kravene 5, 6 eller 9 til 15, hvor det muterede DegP-gen omfatter mutationen S210A.
17. Celle ifølge krav 16, hvor det muterede DegP-gen omfatter SEQ ID NO:9.
18. Celle ifølge et hvilket som helst af de foregående krav, hvor cellen yderligere omfatter en polynukleotidsekvens kodende for et protein af interesse.
19. Celle ifølge krav 18, hvor polynukleotidsekvensen kodende for proteinet af interesse er eksogen.
20. Celle ifølge krav 19, hvor cellen omfatter en ekspressionskassette eller en vektor omfattende polynukleotidsekvensen kodende for proteinet af interesse.
21. Celle ifølge et hvilket som helst af kravene 18 til 20, hvor proteinet af interesse er et antistof eller et antigenbindende fragment deraf.
22. Celle ifølge krav 21, hvor antistoffet eller det antigenbindende fragment deraf er specifikt for TNF.
23. Fremgangsmåde til frembringelse af et rekombinant protein af interesse, hvilken fremgangsmåde omfatter at udtrykke det rekombinante protein af interesse i en rekombinant gramnegativ bakteriecelle som defineret i et hvilket som helst af kravene 1 til 22.
DK15182886.0T 2009-09-24 2010-09-23 Bakteriestamme til rekombinant proteinekspression med proteasefattig DEGP-tilbageholdende chaperoneaktivitet og deaktiverede Tsp- og ptr-gener DK2993231T3 (da)

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Application Number Priority Date Filing Date Title
GBGB0916822.0A GB0916822D0 (en) 2009-09-24 2009-09-24 Bacterial host strain
GB0916821A GB0916821D0 (en) 2009-09-24 2009-09-24 Bacterial host strain
EP10757621A EP2480661A1 (en) 2009-09-24 2010-09-23 Bacterial host strain

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US9315770B2 (en) 2016-04-19
PT2993231T (pt) 2018-11-12
US20150344840A1 (en) 2015-12-03
CY1120947T1 (el) 2019-12-11
IN2012DN02401A (da) 2015-08-21
EP2993231A2 (en) 2016-03-09
BR112012006670B8 (pt) 2022-03-03
SG10201606462WA (en) 2016-09-29
BR112012006670A2 (pt) 2015-09-08
SMT201800549T1 (it) 2018-11-09
WO2011036454A1 (en) 2011-03-31
PL2993231T3 (pl) 2019-01-31
JP6132551B2 (ja) 2017-05-24
LT2993231T (lt) 2018-11-12
AU2016201536A1 (en) 2016-03-24
BR112012006670B1 (pt) 2021-08-03
EP2993231A3 (en) 2016-05-11
KR101758942B1 (ko) 2017-07-17
CA2774692A1 (en) 2011-03-31
HRP20181482T1 (hr) 2018-11-02
KR20120083435A (ko) 2012-07-25
ES2692811T3 (es) 2018-12-05
AU2010299640B2 (en) 2016-02-25
CN102575241B (zh) 2016-02-24
JP2013505714A (ja) 2013-02-21
SI2993231T1 (sl) 2018-10-30
US9550973B2 (en) 2017-01-24
JP2016116517A (ja) 2016-06-30
US9109216B2 (en) 2015-08-18
US20160200810A1 (en) 2016-07-14
AU2010299640A1 (en) 2012-04-12
EP2480661A1 (en) 2012-08-01
CA2774692C (en) 2017-12-19
HK1172650A1 (zh) 2013-04-26
EP2993231B1 (en) 2018-08-01
CN102575241A (zh) 2012-07-11
US20120258492A1 (en) 2012-10-11
HUE040306T2 (hu) 2019-03-28

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