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RU2002105508A - Mutant Enzymes of TN5 Transposase and Method for Their Use - Google Patents

Mutant Enzymes of TN5 Transposase and Method for Their Use

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Publication number
RU2002105508A
RU2002105508A RU2002105508/13A RU2002105508A RU2002105508A RU 2002105508 A RU2002105508 A RU 2002105508A RU 2002105508/13 A RU2002105508/13 A RU 2002105508/13A RU 2002105508 A RU2002105508 A RU 2002105508A RU 2002105508 A RU2002105508 A RU 2002105508A
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Russia
Prior art keywords
transposase
amino acid
mutant
acid sequence
sequence
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RU2002105508/13A
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Russian (ru)
Inventor
Уилль м С. РЕЗНИКОФФ (US)
Уилльям С. РЕЗНИКОФФ
Тодд А. НОМАНН (US)
Тодд А. НОМАНН
Original Assignee
Висконсин Элумни Рисёч Фаундейшн (Us)
Висконсин Элумни Рисёч Фаундейшн
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Publication of RU2002105508A publication Critical patent/RU2002105508A/en

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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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (17)

1. Мутантный протеин Tn5-транспозазы, модифицированный по сравнению с протеином Tn5-транспозазы дикого типа, причем мутантная транспозаза отличается от протеина дикого типа заменой в аминокислотной последовательности, выбранной из группы, включающей аминокислоту в положении 58 и аминокислоту в положении 372, при этом мутантный протеин Tn5-транспозазы обладает предпочтением в отношении внутренних окончаний Tn5 по сравнению с внешними окончаниями Tn5.1. A mutant Tn5 transposase protein modified as compared to a wild-type Tn5 transposase protein, wherein the mutant transposase differs from the wild-type protein by substituting in the amino acid sequence selected from the group consisting of the amino acid at position 58 and the amino acid at position 372, while the mutant the Tn5 transposase protein has preference for the inner ends of Tn5 over the outer ends of Tn5. 2. Мутантная Tn5-транспозаза по п.1, включающая валин в положении 58 аминокислотной последовательности.2. The mutant Tn5 transposase according to claim 1, comprising valine at position 58 of the amino acid sequence. 3. Мутантная Tn5-транспозаза по п.1, включающая мутацию в положении 372 аминокислотной последовательности.3. The mutant Tn5 transposase according to claim 1, comprising a mutation at position 372 of the amino acid sequence. 4. Мутантная Tn5-транспозаза по п.1, включающая глутамин в положении 372 аминокислотной последовательности.4. The mutant Tn5 transposase according to claim 1, comprising glutamine at position 372 of the amino acid sequence. 5. Мутантная Tn5-транспозаза по п.1, где мутантная транспозаза отличается от протеина дикого типа аминокислотами в положениях 58 и 372.5. The mutant Tn5 transposase according to claim 1, wherein the mutant transposase is different from the wild-type protein by amino acids at positions 58 and 372. 6. Мутантная Tn5-транспозаза по п.1, включающая валин в положении 58 аминокислотной последовательности и глутамин в положении 372 аминокислотной последовательности.6. The mutant Tn5 transposase according to claim 1, comprising valine at position 58 of the amino acid sequence and glutamine at position 372 of the amino acid sequence. 7. Мутантная Tn5-транспозаза по п.1, имеющая дополнительные отличия от Tn5-транспозазы дикого типа, представляющие собой замены в аминокислотной последовательности, выбранные из группы, включающей аминокислоту в положении 8 и аминокислоту в положении 344.7. The mutant Tn5 transposase according to claim 1, having additional differences from wild-type Tn5 transposase, which are substitutions in the amino acid sequence selected from the group consisting of the amino acid at position 8 and the amino acid at position 344. 8. Мутантная Tn5 -транспозаза по п.7, в которой аминокислота в положении 8 представляет собой цистеин.8. The mutant Tn5 transposase of claim 7, wherein the amino acid at position 8 is cysteine. 9. Мутантная Tn5-транспозаза по п.7, в которой аминокислота в положении 344 представляет собой лизин.9. The mutant Tn5 transposase according to claim 7, wherein the amino acid at position 344 is lysine. 10. Мутантная Tn5-транспозаза по п.7, где мутантная Tn5-транспозаза отличается от Tn5-транспозазы дикого типа заменой аминокислоты в положении 8 и аминокислоты в положении 344.10. The mutant Tn5 transposase according to claim 7, wherein the mutant Tn5 transposase is different from wild-type Tn5 transposase by replacing the amino acid at position 8 and the amino acid at position 344. 11. Мутантная Tn5-транспозаза по п.1, включающая цистеин в положении 8 аминокислотной последовательности и лизин в положении 344 аминокислотной последовательности.11. The mutant Tn5 transposase according to claim 1, comprising cysteine at position 8 of the amino acid sequence and lysine at position 344 of the amino acid sequence. 12. Мутантная Tn5-транспозаза по п.1, включающая цистеин в положении 8 аминокислотной последовательности, валин в положении 58 аминокислотной последовательности, лизин в положении 344 аминокислотной последовательности и глутамин в положении 372 аминокислотной последовательности.12. The mutant Tn5 transposase according to claim 1, comprising cysteine at position 8 of the amino acid sequence, valine at position 58 of the amino acid sequence, lysine at position 344 of the amino acid sequence and glutamine at position 372 of the amino acid sequence. 13. Полинуклеотид, который кодирует транспозазу по п.1.13. Polynucleotide that encodes a transposase according to claim 1. 14. Полинуклеотид, который кодирует транспозазу по п.12.14. A polynucleotide that encodes a transposase according to claim 12. 15. Система для транспозиции мобильной последовательности ДНК in vitro, которая включает: мутантную Tn5-транспозазу по п.1, модифицированную по сравнению с Tn5-транспозазой дикого типа, молекулу ДНК-донора, включающую мобильную последовательность ДНК, причем последовательность фланкирована на 5'- и 3'-концах последовательностью IE дикого типа, и молекулу ДНК-мишени, в которую мобильный элемент может быть перенесен.15. A system for transposition of a mobile DNA sequence in vitro, which includes: the mutant Tn5 transposase according to claim 1, modified in comparison with wild-type Tn5 transposase, a donor DNA molecule comprising a mobile DNA sequence, the sequence being flanked at 5'- and the 3'-ends of the wild-type IE sequence, and a target DNA molecule into which the mobile element can be transferred. 16. Система по п.15, где последовательность IE является метилированной.16. The system of claim 15, wherein the IE sequence is methylated. 17. Способ транспозиции in vitro, предусматривающий следующие стадии: объединение молекулы ДНК-донора, которая включает представляющую интерес мобильную последовательность ДНК, при этом представляющая интерес последовательность ДНК фланкирована на 5'- и 3'-концах последовательностью IE Tn5 дикого типа, с молекулой ДНК-мишенью и мутантной Tn5-транспозазой по п.1, в приемлемом реакционном буфере при температуре ниже физиологической температуры до тех пор, пока модифицированная транспозаза не свяжется с последовательностями IE, и повышение температуры до физиологической температуры в течение периода времени, достаточного для того, чтобы фермент катализировал транспозицию in vitro.17. An in vitro transposition method comprising the steps of: combining a donor DNA molecule that comprises a mobile DNA sequence of interest, the DNA sequence of interest being flanked at the 5 ′ and 3 ′ ends of the wild-type IE Tn5 sequence with a DNA molecule the target and the mutant Tn5 transposase according to claim 1, in an acceptable reaction buffer at a temperature below physiological temperature until the modified transposase binds to IE sequences, and the temperature increases ry to a physiological temperature for a period of time sufficient for the enzyme to catalyze transposition in vitro.
RU2002105508/13A 1999-08-02 2000-08-02 Mutant Enzymes of TN5 Transposase and Method for Their Use RU2002105508A (en)

Applications Claiming Priority (2)

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US14668699P 1999-08-02 1999-08-02
US60/146,686 1999-08-02

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JP (1) JP2003505104A (en)
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CA (1) CA2380850A1 (en)
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CA2380850A1 (en) 2001-02-08
WO2001009363A1 (en) 2001-02-08
CN1367840A (en) 2002-09-04
AU775043B2 (en) 2004-07-15
EP1198583A1 (en) 2002-04-24
AU6396700A (en) 2001-02-19
JP2003505104A (en) 2003-02-12
PL353241A1 (en) 2003-11-03

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