DK2297333T3 - Fremgangsmåde til rumlig adskillelse og til screening af celler - Google Patents

Fremgangsmåde til rumlig adskillelse og til screening af celler Download PDF

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DK2297333T3
DK2297333T3 DK09755288.9T DK09755288T DK2297333T3 DK 2297333 T3 DK2297333 T3 DK 2297333T3 DK 09755288 T DK09755288 T DK 09755288T DK 2297333 T3 DK2297333 T3 DK 2297333T3
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enzyme
cells
activity
library
enzymes
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DK09755288.9T
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Kerry Love
J Christopher Love
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Massachusetts Inst Technology
Whitehead Biomedical Inst
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
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  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Claims (16)

1. Fremgangsmåde til at gennemføre en screening af enzymer i opløsningsfase, omfattende: afsætte et bibliotek af celler på en mikroindretning, hvor mikroindretningen indeholder brønde, som rumligt adskiller cellerne i opløsning, hvor cellerne fordeles med én celle per brønd i gennemsnit, hvor en flerhed af cellerne sekrerer varianter af mindst én enzym i opløsningen; bringe de sekrerede enzymvarianter i opløsningen i kontakt med mindst ét optisk signalsubstrat; hvor enzymaktiviteten overvåges ved at detektere ændringer over tiden i et eller flere optiske signaler, som genereres af det ene eller flere optiske signalsubstrater i cellebiblioteket, hvor sådanne ændringer angiver en ønsket aktivitet af varianterne af enzymet.
2. Fremgangsmåde ifølge krav 1, hvor det optiske signal er et fluorescenssignal.
3. Fremgangsmåde ifølge krav 2, hvor enzymaktiviteten overvåges i realtid eller næsten realtid i mikroindretningen på basis af ændringer i intensiteterne af fluorescerenssignal.
4. Fremgangsmåde ifølge krav 1, hvor enzymet er udvalgt fra gruppen bestående af protease, oxidoreduktase, transferase, hydrolase, lyase, isomera-se og ligase.
5. Fremgangsmåde ifølge krav 4, hvor molekylvægten af enzymet er større end ca. 600 Da og mindre end ca. 100.000 Da.
6. Fremgangsmåde ifølge krav 1, hvor enzymaktiviteten vurderes på basis af flere parametre, der er udvalgt fra gruppen bestående af katalytisk hastighed, reaktionsspecificitet, kinetisk effektivitet og substratbindingsaffinitet.
7. Fremgangsmåde ifølge krav 6, hvor parametrene vurderes parallelt.
8. Fremgangsmåde ifølge krav 1, hvor cellerne er eukaryotiske celler.
9. Fremgangsmåde ifølge krav 8, hvor de eukaryotiske celler er gærceller.
10. Fremgangsmåde ifølge krav 1, hvor brøndene er mellem ca. 10 og ca. 100 pm i diameter.
11. Fremgangsmåde ifølge krav 1, yderligere omfattende generering af varianterne med en trin, som omfatter mutation af det gen, som koder for enzymet.
12. Fremgangsmåde ifølge krav 1, yderligere omfattende genvinding af de celler, som sekrerer en ønsket enzymvariant fra mikroindretningen.
13. Fremgangsmåde ifølge krav 12, hvor genvindingen sker ved hjælp af mi-kromanipulation med glaskapillærer.
14. Fremgangsmåde ifølge krav 13, yderligere omfattende isolering og sekvensering af DNA, som koder for den ønskede enzymvariant.
15. Fremgangsmåde ifølge krav 2, hvor enzymet er en mutation af glycosyl-transferase (GTase) eller glycosidase.
16. Fremgangsmåde ifølge krav 15, hvor GTase er i stand til at konkurrere med kemisk syntese om hurtig fremstilling i stor målestok af komplekse kulhydrater.
DK09755288.9T 2008-05-30 2009-06-01 Fremgangsmåde til rumlig adskillelse og til screening af celler DK2297333T3 (da)

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US5737108P 2008-05-30 2008-05-30
PCT/US2009/003354 WO2009145925A1 (en) 2008-05-30 2009-06-01 Compositions and methods for spatial separation and screening of cells

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EP (1) EP2297333B1 (da)
JP (1) JP5718221B2 (da)
CN (1) CN102112624A (da)
CA (1) CA2763790C (da)
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US20180335419A1 (en) 2018-11-22
CA2763790A1 (en) 2009-12-03
JP5718221B2 (ja) 2015-05-13
JP2011521644A (ja) 2011-07-28
US20210102933A1 (en) 2021-04-08
EP2297333A4 (en) 2012-03-14
CN102112624A (zh) 2011-06-29
EP2297333A1 (en) 2011-03-23
US20110124520A1 (en) 2011-05-26
CA2763790C (en) 2016-11-22
WO2009145925A1 (en) 2009-12-03
EP2297333B1 (en) 2015-01-28

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