DK2990478T3 - Syntetiske oplophorus-luciferaser med forbedret lysemission - Google Patents
Syntetiske oplophorus-luciferaser med forbedret lysemission Download PDFInfo
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- DK2990478T3 DK2990478T3 DK15186652.2T DK15186652T DK2990478T3 DK 2990478 T3 DK2990478 T3 DK 2990478T3 DK 15186652 T DK15186652 T DK 15186652T DK 2990478 T3 DK2990478 T3 DK 2990478T3
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- Prior art keywords
- gly
- val
- leu
- lys
- thr
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Classifications
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- 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
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
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Claims (19)
1. Polypeptid, der har mindst 60 % aminosyresekvensidentitet med en vildtype Oplophorus-\uc\ferase, og der omfatter mindst en aminosyresubstitution ved en position valgt fra positionerne 2, 4, 11, 20, 23, 28, 33, 34, 44, 45, 51, 54, 68, 72, 75, 76, 77, 89, 90, 92, 99, 104, 115, 124, 135, 138, 139, 143, 144, 164, 166, 167, eller 169 svarende til SEQ ID NO:l, hvor polypeptidet har mindst en af forbedret luminescens, forbedret signalstabilitet, og forbedret proteinstabilitet i forhold til vildtype Op/opftorus-luciferasen.
2. Polypeptidet ifølge krav 1, hvor polypeptidet har forbedret luminescens i forhold til vildtype Oplophorus-\uc\feraseu og en aminosyresubstitution ved én eller flere af positionerne 4, 11, 20, 28, 33, 54, 68, 75, 115, 124, 138, 143, eller 166 svarende til SEQ ID NO:l.
3. Polypeptidet ifølge et hvilket som helst af de foregående krav, hvor polypeptidet har forbedret proteinstabilitet i forhold til vildtype Oplophorus-luciferase, og en aminosyresubstitution ved én eller flere af positionerne 11, 20, 28, 33, 34, 44, 45, 51, 54, 68, 72, 75, 77, 89, 90, 92, 99, 104, 115, 124, 135, 138, 139, 143, 144, 164, 166, 167, eller 169 svarende til SEQ ID NO:l.
4. Polypeptidet ifølge et hvilket som helst af kravene 1-3, hvor polypeptidet yderligere omfatter en deletion af luciferaserester svarende til N-terminus af vildtype Op/opPorus-luciferasen.
5. Polypeptidet ifølge krav 4, hvor deletionen er 27 eller færre rester af N-terminus af vildtype Oplophorus-luciferasesekvensen.
6. Polypeptidet ifølge et hvilket som helst af kravene 1-5, hvor vildtype Oplophorus-\uc\ferasen er fra Oplophorus gracilirostris, Oplophorus grimaldii, Oplophorus spinicauda, Oplophorus foliaceus, Oplophorus novaezelandiae, Oplophorus typus, eller Oplophorus spinous.
7. Polypeptidet ifølge et hvilket som helst af kravene 1-6, hvor polypeptidet omfatter en substitution svarende til mindst én af T2S, A4E/S/R/G/D/T/L, Q11R/V/I/L/K/T, Q20R, E23V, S28P, A33K, L34M, V44I/L, V45E, G51V, A54F/T/V/G/S/W/L/I, F68S/Y/V, L72Q, M75R/K/Q/G/T/A, I76V, F77W, V79I, K89E, I90T/V, L92S/A/G/Q, I99V, P104L, P115E/I/Q/L/V/G/H/R/S/C/A/T, Q124K, N135K, Y138V/I/N/T/UC/R/M/K, D139E, I143L, N144K, C164S, N166R/K/A/L/P/Q/S, 1167V, eller 169L svarende til SEQ ID NO:l.
8. Polypeptidet ifølge et hvilket som helst af kravene 1-7, hvor polypeptidet omfatter aminosyresubstitutioner: (i) ved positionerne 11, 33, 44, 54, 115, 124, 138, og 166 svarende til SEQ ID NO: 1, (ii) ved positionerne 23, 28, 143, og 166 svarende til SEQ ID NO:l, (iii) ved positionerne 4, 34, 76, og 166 svarende til SEQ ID NO: 1, eller (iv) ved positionerne 51, 99, og 166 svarende til SEQ ID NO:l.
9. Polypeptidet ifølge krav 8, hvor polypeptidet yderligere omfatter aminosyresubstitutioner ved positionerne 45, 135, 104, 139, og 167 svarende til SEQ ID NO: 1.
10. Polypeptidet ifølge krav 8 eller 9, hvor polypeptidet yderligere omfatter aminosyresubstitutioner ved positionerne 28, 34, 51, 99 og 143 svarende til SEQ ID NO: 1.
11. Polypeptidet ifølge et af kravene 8-10, yderligere omfattende en substitution af mindst én aminosyre ved position 20, 54, 68, 72, 77, 79, 89, 90, 92, eller 164 svarende til SEQ ID NO:l.
12. Polypeptidet ifølge et hvilket som helst af kravene 1-11, hvor polypeptidet er et modificeret luciferasepolypeptid.
13. Fusionsprotein omfattende polypeptidet ifølge et af kravene 1-12 forbundet til et polypeptid af interesse.
14. Polynukleotid, der koder for fusionsproteinet ifølge krav 13.
15. Vektor omfattende polynukleotidet ifølge krav 14.
16. Vektoren ifølge krav 15, hvor polynukleotidet er operativt forbundet til en promoter.
17. Celle omfattende polypeptidet ifølge et af kravene 1-12, fusionsproteinet ifølge krav 13, polynukleotidet ifølge krav 14, eller vektoren ifølge krav 15 eller 16.
18. Ikke-humant transgent dyr omfattende polynukleotidet ifølge krav 14, vektoren ifølge krav 15 eller 16, eller cellen ifølge krav 17.
19. Kit omfattende polypeptidet ifølge et af kravene 1-12, fusionsproteinet ifølge krav 13, polynukleotidet ifølge krav 14, vektoren ifølge krav 15 eller 16, eller cellen ifølge krav 17.
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