DK200401821A - Fremgangsmåde til in vitro molekylær udvikling af proteinfunktion - Google Patents
Fremgangsmåde til in vitro molekylær udvikling af proteinfunktion Download PDFInfo
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- DK200401821A DK200401821A DK200401821A DKPA200401821A DK200401821A DK 200401821 A DK200401821 A DK 200401821A DK 200401821 A DK200401821 A DK 200401821A DK PA200401821 A DKPA200401821 A DK PA200401821A DK 200401821 A DK200401821 A DK 200401821A
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- 238000000034 method Methods 0.000 title abstract description 39
- 238000000338 in vitro Methods 0.000 title abstract 2
- 230000004853 protein function Effects 0.000 title abstract 2
- 239000002157 polynucleotide Substances 0.000 abstract description 76
- 102000040430 polynucleotide Human genes 0.000 abstract description 76
- 108091033319 polynucleotide Proteins 0.000 abstract description 76
- 108060002716 Exonuclease Proteins 0.000 abstract description 17
- 102000013165 exonuclease Human genes 0.000 abstract description 17
- 239000011541 reaction mixture Substances 0.000 abstract description 14
- 239000012634 fragment Substances 0.000 abstract description 12
- 230000029087 digestion Effects 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 239000000203 mixture Substances 0.000 abstract description 4
- 238000000137 annealing Methods 0.000 abstract 1
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 108090000765 processed proteins & peptides Proteins 0.000 description 15
- 239000003814 drug Substances 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 1
- 108010046914 Exodeoxyribonuclease V Proteins 0.000 description 1
- 102100029075 Exonuclease 1 Human genes 0.000 description 1
- 102000019236 Exonuclease V Human genes 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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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
- 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
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
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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
- 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
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Ecology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
reaktionsvolumenet, varigheden af fordøjelsesreaktionen, temperaturen af reaktionsblandingen, pH af reaktionsblandingen, længden af de enkeltstrengede moder-polynukleotidsekvenser, mængden af enkeltstrengede polynukleotidmolekyler og puffersammensætningen af reaktionsblandingen.
PATENTKRAV 1. Fremgangsmåde til frembringelse af en polynukleotidsekvens eller population af sekvenser ud fra enkeltstrengede moderpolynukleotidsekvenser, og som indkoder én eller flere proteinmotiver, idet fremgangsmåden omfatter trinnene, hvor man a) tiivejebringer en første population af enkeltstrengede polynukleotid-molekyler og en nummer to population af enkeltstrengede polynukleotid-molekyier, idet den første og nummer to populationen tilsammen udgør plus- og minusstrenge af moderpolynukleotidsekvenser; b) udfører en reaktion til fordøjelse af den første og nummer to populationen af enkeltstrengede polynukleotidmolekyler med en exonuklease for at frembringe tilsvarende populationer af enkeltstrengede polynukleotidfrag-menter; c) bringer de polynukleotidfragmenter, der er frembragt fra plusstrengene, i berøring med fragmenter, der er frembragt fra minusstrengene; og d) formerer de fragmenter, som hybridiserer til hinanden for at frembringe i det mindste én polynukleotidsekvens, der indkoder én eller flere protein-motiver, som har ændrede egenskaber sammenlignet med det eller de proteinmotiver, som indkodes af moderpolynukleotiderne, hvorved der i trin b) i det mindste er én parameter i den reaktion, der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, der er forskellig fra den eller de ækvivalente parametre, der anvendes i reaktionen til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 2. Fremgangsmåde ifølge krav 1, hvorved reaktionsparameteren vælges blandt exonukleasetypen, exonukleasekoncentrationen, reaktionsvolumenet, varigheden af fordøjelsesreaktionen, temperaturen af reaktionsblandingen, pH af reaktionsblandingen, længden af de enkeltstrengede moderpolynukleotidsekvenser, mængden af enkeltstrengede polynukleotidmolekyler og puffersammensætningen af reaktionsblandingen. 3. Fremgangsmåde ifølge krav 1 eller 2, hvorved den exonuklease, der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er forskellig fra den exonuklease, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 4. Fremgangsmåde ifølge krav 3, hvorved den exonuklase, der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er en 3'-exo-nuklease og den exonuklease, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler er en 5'-exonuklease. 5. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved den exonukleasekoncentration der anvendes til fordøjelse af den første population af enkeitstrengede polynukleotidmolekyler, er forskellig fra den exonukleasekoncentration, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 6. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved det reaktionsvolumen, der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er forskellig fra det reaktionsvolumen, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 7. Fremgangsmåde ifølge et hvilket som helst af de fregående krav, hvorved varigheden af den fordøjelsesreaktion, der anvendes til fordøjelsen af den første population af enkeltstrengede polynukleotidmolekyler. er forskellig fra varigheden af den fordøjelsesreaktion, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 8. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved temperaturen af den reaktionsblanding der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er forskellig fra temperaturen af den reaktionsblanding, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 9. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved pH af den reaktionsblanding, der anvendes ti! fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er forskellig fra pH-værdien af den reaktions blanding, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 10. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved længden af polynukleotiderne i den første population af enkeltstrengede polynukleotidmolekyler er forskellig fra længden af polynukleotiderne i nummer to populationen af enkeltstrengede polynukleotidmolekyler. 11. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved puffersammensætningen af den reaktionsblanding, der anvendes til fordøjelse af den første population af enkeltstrengede polynukleotidmolekyler, er forskellig fra puffer-sammensætningen af den reaktionsblanding, der anvendes til fordøjelse af nummer to populationen af enkeltstrengede polynukleotidmolekyler. 12. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved mængden af enkeltstrengede polynukleotidmolekyler i den første population af enkeltstrengede polynukleotidmolekyler er forskellig fra mængden af enkeltstrengede polynukleotidmolekyler i nummer to populationen af enkeltstrengede polynukleotidmolekyler. 13. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved den første population af enkeltstrengede polynukleotidmolekyler udgør plusstrenge af moderpolynukleotidsekvenser, og nummer to populationen af enkeltstrengede polynukleotidmolekyler udgør minusstrenge af moderpolynukleotidsekvenser, 14. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved polynukleotidmolekylerne i trin a) er DNA-molekyler. 15. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved trin c) yderligere omfatter, at man tilsætter primersekvenser, som hybridisere til 3'- og/eller 5'-enderne af i det mindste et af moderpolynukleotiderne under hybridiseringsbetingelser. 16. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved den exonuklease, der anvendes til at fordøje den første og/eller nummre to populationen af enkeltstrengede polynukleotidmolekyler er valgt fra gruppen bestående af BAL31,
Exonuklease I, Exonuklease V, Exonuklease VII, Exonuklease T7gen6, bakteriofag lambda exonuklease og Exonuklease Rec Jf. 17. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved en moderpolynukleotidsekvens eller -sekvenser underkastes mutagenese. 18. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved én eller begge af populationen af fragmenter, der er frembragt i trin b), underkastes mutagenese. 19. Fremgangsmåde ifølge krav 17 eller 18, hvorved mutagenesen er fejltilbøjelig PCR. 20. Fremgangsmåde ifølge et hvilket som helst af de foreågende krav, hvorved trin b) udføres for at frembringe populationer af enkeltstrengede fragmenter med varierende længder. 21. Fremgangsmåde ifølge krav 20, hvorved trin b) reguleres for at frembringe en population af enkeltstrengede fragmenter med en genemsnitiig længde på mere end omkring 50 nukleotider. 22. Fremgangsmåde ifølge et hvilket som helst af de foregående krav og som yderligere omfatter det trin, hvor man eksprimerer i det mindste én polynukleo-tidsekvens, der er frembragt i trin d), for at frembringe det indkodede polypeptid. 23. Fremgangsmåde ifølge krav 22 og som yderligere omfatter trinnet, hvor man tester det indkodede polypeptid for ønskede egenskaber. 24. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved moderpolynukleotidsekvensen indkoder et antistof eller fragment deraf. 25. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved moderpolynukleotidsekvensen indkoder et enzym. 26. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved moderpolynukleotidsekvensen indkoder et antigen. 27. Fremgangsmåde til fremstilling af et polypeptid med ønskede egenskaber, idet fremgangsmåden omfatter de følgende trin: a) frembringelse af forskellige former af et moderpolynukleotid ved anvendelse af en fremgangsmåde ifølge et hvilket som helst af kravene 1 til 26; b) eksprimerer de forskellige polynukleotider, der er frembragt i trin a), for at frembringe forskellige polypeptider; c) screening af de forskellige polypeptider for ønskede egenskaber; og d) udvælgelse af et polypeptid med ønskede egenskaber fra de forskellige polypeptider. 28. Polypeptid opnået ved en fremgangsmåde ifølge krav 27. 29. Farmaceutisk sammensætning omfattende et polypeptid ifølge krav 28 og en farmaceutisk acceptabel bærer. 30. Polypeptid ifølge krav 28 til anvendelse i medicin. 31. Anvendelse af et polypeptid ifølge krav 28 i fremstillingen af et lægemiddel til behandling, terapi og/eller diagnose afen sygdom. 32. Fremgangsmåde til fremstilling af en farmaceutisk sammensætning, hvilken fremgangsmåde omfatter, at man efter identifikationen af et polynukleotid og/eller et indkodet polypeptid med ønskede egenskaber ved en fremgangsmåde ifølge et hvilket som helst af kravene 1 til 26, tilsætter polynukleotidet og/eller det indkodede polypeptid til en farmaceutisk acceptabel bærer. 33. Fremgangsmåde som omfatter, at man efter identifikation af et polynukleotid og/eller et indkodet polypeptid med ønskede egenskaber ved en fremgangsmåde ifølge et hvilket som helst af kravene 1 til 26, anvender dette polynukleotid og/eller indkodede polypeptid helt eller delvis inden for medicinen. 34. Fremgangsmåpde ifølge krav 33, hvorved anvendelsen inde for medicinen er i behandlingen, terapien og/eller diagnosen af en sygdom. 35. Fremgangsmåde som omfatter, at man efter identifikation af et polynukleotid med ønskede egenskaber ifølge en fremgangsmåde ifølge et hvilket som helst af kravene 1 til 26, anvender dette polynukleotid til påvisning af og/eller formering af et målpolynukleotid i en prøve.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0211369A GB2388604B (en) | 2002-05-17 | 2002-05-17 | A method for in vitro molecular evolution of protein function |
| US10/321,195 US7153655B2 (en) | 1998-06-16 | 2002-12-17 | Method for in vitro molecular evolution of protein function involving the use of exonuclease enzyme and two populations of parent polynucleotide sequence |
| PCT/GB2003/002102 WO2003097834A2 (en) | 2002-05-17 | 2003-05-16 | A method for in vitro molecular evolution of protein function |
Publications (1)
| Publication Number | Publication Date |
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| DK200401821A true DK200401821A (da) | 2004-11-23 |
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Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK03722867.3T DK1504098T4 (da) | 2002-05-17 | 2003-05-16 | Fremgangsmåde til molekyulær udvikling in vitro af proteinfunktion |
| DK200401821A DK200401821A (da) | 2002-05-17 | 2004-11-23 | Fremgangsmåde til in vitro molekylær udvikling af proteinfunktion |
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| Application Number | Title | Priority Date | Filing Date |
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| DK03722867.3T DK1504098T4 (da) | 2002-05-17 | 2003-05-16 | Fremgangsmåde til molekyulær udvikling in vitro af proteinfunktion |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US7262012B2 (da) |
| EP (1) | EP1504098B2 (da) |
| JP (1) | JP4372679B2 (da) |
| AT (1) | ATE356871T1 (da) |
| AU (1) | AU2003230027A1 (da) |
| CA (1) | CA2485506C (da) |
| DE (1) | DE60312507T3 (da) |
| DK (2) | DK1504098T4 (da) |
| ES (1) | ES2285118T5 (da) |
| WO (1) | WO2003097834A2 (da) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309662A (zh) * | 1998-07-10 | 2001-08-22 | 亚利制药有限公司 | 葡萄球菌属的趋化性抑制蛋白(chips)及其应用 |
| EP1118663A1 (en) * | 2000-01-07 | 2001-07-25 | Universiteit Utrecht | Nucleic acids encoding chemotaxis inhibitory polypeptides |
| US20020086292A1 (en) | 2000-12-22 | 2002-07-04 | Shigeaki Harayama | Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules |
| EP1586583A3 (en) * | 2004-04-16 | 2005-11-16 | Alligator Bioscience AB (publ) | Compounds that block C5a complement receptor and their use in therapy |
| GB2432366B (en) * | 2005-11-19 | 2007-11-21 | Alligator Bioscience Ab | A method for in vitro molecular evolution of protein function |
| GB0607798D0 (en) | 2006-04-20 | 2006-05-31 | Alligator Bioscience Ab | Novel polypeptides and use thereof |
| GB0708376D0 (en) | 2007-05-01 | 2007-06-06 | Alligator Bioscience Ab | Novel polypeptides and uses thereof |
| GB0905790D0 (en) | 2009-04-03 | 2009-05-20 | Alligator Bioscience Ab | Novel polypeptides and use thereof |
| GB0905503D0 (en) * | 2009-03-31 | 2009-05-13 | Alligator Bioscience Ab | A method for in vitro molecular evolution of protein function |
| GB0920258D0 (en) | 2009-11-19 | 2010-01-06 | Alligator Bioscience Ab | New medical agents and use thereof |
| GB201019086D0 (en) | 2010-11-11 | 2010-12-29 | Imp Innovations Ltd | Bacterial methods |
| US20140178921A1 (en) | 2011-03-04 | 2014-06-26 | Evolvemol, Inc. | Apparatus and process for isolating specific physical items within a set of physical items |
| GB201115280D0 (en) | 2011-09-05 | 2011-10-19 | Alligator Bioscience Ab | Antibodies, uses and methods |
| GB201311475D0 (en) | 2013-06-27 | 2013-08-14 | Alligator Bioscience Ab | Polypeptides |
| CA2957146A1 (en) | 2014-08-12 | 2016-02-18 | Alligator Bioscience Ab | Combination therapies with anti cd40 antibodies |
| BR112018006251A2 (pt) | 2015-09-30 | 2018-10-16 | Janssen Biotech Inc | anticorpos antagonistas que se ligam especificamente a cd40 humano e métodos de uso |
| CN113892654A (zh) * | 2021-10-21 | 2022-01-07 | 长春大学 | 一种以绿豆蛋白粉为原料制备免消化绿豆蛋白质的方法 |
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| EP1504098A2 (en) | 2005-02-09 |
| CA2485506C (en) | 2012-02-28 |
| AU2003230027A1 (en) | 2003-12-02 |
| DE60312507D1 (de) | 2007-04-26 |
| US20060166198A1 (en) | 2006-07-27 |
| CA2485506A1 (en) | 2003-11-27 |
| JP2005525820A (ja) | 2005-09-02 |
| DK1504098T4 (da) | 2011-05-23 |
| EP1504098B2 (en) | 2011-02-09 |
| AU2003230027A8 (en) | 2003-12-02 |
| DE60312507T2 (de) | 2007-12-06 |
| DK1504098T3 (da) | 2007-06-11 |
| ES2285118T3 (es) | 2007-11-16 |
| DE60312507T3 (de) | 2011-08-18 |
| ATE356871T1 (de) | 2007-04-15 |
| EP1504098B1 (en) | 2007-03-14 |
| WO2003097834A3 (en) | 2004-02-19 |
| WO2003097834A2 (en) | 2003-11-27 |
| US7262012B2 (en) | 2007-08-28 |
| JP4372679B2 (ja) | 2009-11-25 |
| ES2285118T5 (es) | 2012-09-21 |
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