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Mutant tRNA for introducing unnatural amino acid into protein 実績あり

外国特許コード F120006773
整理番号 K01709WO
掲載日 2012年6月18日
出願国 アメリカ合衆国
出願番号 08496706
公報番号 20110224411
公報番号 8372960
出願日 平成18年11月14日(2006.11.14)
公報発行日 平成23年9月15日(2011.9.15)
公報発行日 平成25年2月12日(2013.2.12)
国際出願番号 JP2006323064
国際公開番号 WO2007055429
国際出願日 平成18年11月14日(2006.11.14)
国際公開日 平成19年5月18日(2007.5.18)
優先権データ
  • 特願2005-329115 (2005.11.14) JP
  • 2006JP323064 (2006.11.14) WO
発明の名称 (英語) Mutant tRNA for introducing unnatural amino acid into protein 実績あり
発明の概要(英語) It is an objective of the present invention to provide tRNA that has CUA or CCCG as an anticodon and is aminoacylated with an unnatural amino acid, such tRNA being capable of efficiently introducing an unnatural amino acid into a protein without competing with a termination factor.
Such tRNA is a mutant tRNA for tryptophan which has G at the 5′ end, C as a base pairing with the G at the 5′ end, and A as a base next to the C on the 3′ side, such tRNA being a mutant tRNA which pairs with a stop codon and has CUA as an anticodon or a mutant tRNA which pairs with a stop codon or a 4-base codon has CUA or CCCG as an anticodon, into which a single base has been inserted just before the CCA sequence at the 3′ end.
従来技術、競合技術の概要(英語) BACKGROUND ART
In general, chemical modification of a specific protein residue is carried out to introduce a functional group onto the surface of a protein.
Such chemical modification can be readily carried out and many specific residues can be modified at once, which is advantageous.
On the other hand, excellent results are unlikely to be obtained in terms of reproducibility of the control of modified sites and/or the number of modified sites, which is problematic.
Along with the recent developments in genetic engineering, it has become possible to substitute amino acid residues in proteins.
Thus, it has become possible to introduce a desired unnatural amino acid having an amino skeleton into a protein by modifying a protein synthesis system, resulting in the realization of synthesis of a protein carrying functional groups with good reproducibility.
During protein synthesis, an amino acid first binds to the 3' end of tRNA and then is transferred to a ribosome, where protein synthesis takes place.
In a ribosome, translation from codons to amino acids takes place.
With the use of tRNA bound to an unnatural amino acid, an unnatural amino acid can be incorporated into a protein.
The following method can be used for introducing an unnatural amino acid into a protein: a method wherein a codon at a target site for introduction is first substituted with a stop codon UAG and then translation is carried out in the presence of tRNA that has CUA as an anticodon and is aminoacylated with an unnatural amino acid (see Non-Patent Documents 1 to 4).
In such a method, examples of tRNA used include tRNA for yeast phenylalanine (see Non-Patent Documents 1 and 2), tRNA for E. coli asparagine, tRNA for tetrahymena glutamine (see Non-Patent Document 3), and tRNA for E. coli glycine (see Non-Patent Document 4).
However, since tRNA that has CUA as an anticodon and is aminoacylated with an unnatural amino acid competes with a termination factor upon translation of UAG, the efficiency of introduction of an unnatural amino acid is not high in such case.
Non-Patent Document 1: Science, 244, p. 182, 1989
Non-Patent Document 2: Nucleic Acids Res., 18, 83-88, 1989
Non-Patent Document 3: Chem.
Biol., 3, 1033-1038, 1996
Non-Patent Document 4: J. Am. Chem. Soc., 111, p. 8013, 1989

特許請求の範囲(英語) [claim1]
1. A mutant tRNA, which is a mutant of tRNA for tryptophan obtained from a microorganism selected from the group consisting of Mycoplasma capricolum, Bacillus halodouranns, Bacillus subtillis, Borrelia burgdorferi, Mycoplasma genitalium, Mycoplasma pneumoniae 1, Mycoplasma pneumoniae 2 and Staphylococcus aureus N315, wherein the mutation consists of;
(i) G at the 5' end,
(ii) C at 5th base from the 3' end which pairs with the G of (i) at the 5' end, and A at 4th base from the 3' end which is adjacent to the 3' side of the C of (ii),
wherein the mutant tryptophan tRNA (mtRNAcuACUAtrp) pairs with a UAG codon and has CUA as an anticodon; and
where said mtRNACUAtrp has a higher efficiency of incorporation of an unnatural amino acid, a modified amino acid or a derivative thereof into a protein in an in-vitro cell-free translation system compared to the wild type tRNAtrp or to a tRNACUAtrP that does not comprise the mutations of (i), (ii) or (iii).
[claim2]
2. The mutant tRNA of claim 1, which consists of nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:25.
[claim3]
3. The mutant tRNA of claim 1 into which a single base A, C, G, or U has been inserted at the 4th position from the 3' end, which is adjacent to 5' side of ACC sequence located at 1st to 3rd position from the 3' end.
[claim4]
4. The mutant tRNA of claim 1, which is aminoacylated with an amino acid derivative and wherein the amino acid derivative is selected from the group consisting of hydroxy acid, mercapto acid, and carboxylic acid.
[claim5]
5. The mutant tRNA of claim 1, wherein the amino acid is fluorescently-labeled.
[claim6]
6. A method for introducing an amino acid selected from the group consisting of an unnatural amino acid, a modified amino acid, or a derivative thereof into a protein, the method comprising: providing an mRNA of a protein into which said amino acid is introduced; the mutant tRNA of claim 1 and
allowing the mutant tRNA to pair with the UAG codon, wherein the mRNA has a UAG codon that is a codon corresponding to a site at which an amino acid is introduced thereby introducing said amino acid into the protein.
[claim7]
7. A method of producing a protein comprising two different phosphor amino acids in an in-vitro translation system, wherein the method comprises: preparing an mRNA into which a single 4-base codon and a single UAG codon have been inserted at specified positions;
preparing two mutant tRNA molecules of claim 1 comprising anti-codons that pair with the 4-base codon and the UAG codon, and wherein one of the tRNA molecules is bound to an amino acid labeled with the fluorescent energy donor and the other tRNA is bound to an amino acid labeled with a fluorescent energy acceptor; and
allowing the translation system to synthesize the protein wherein, the protein encoded by said mRNA displays a change in distance and orientation upon interaction with other molecules and results in a change in the efficiency of fluorescence resonance energy transfer.
[claim8]
8. The method of claim 6, wherein protein synthesis is carried out in a cell-free translation system.
[claim9]
9. The method of claim 7, wherein protein synthesis is carried out in a cell-free translation system.
[claim10]
10. A method for introducing an amino acid selected from the group consisting of an unnatural amino acid, a modified amino acid, or a derivative thereof into a protein, the method comprising: introducing an amino acid into a protein in a manner such that mRNA of a protein into which an amino acid is introduced and the mutant tRNA of claim 3 are employed; and
allowing the mutant tRNA to pair with the UAG codon, wherein the mRNA has a UAG codon that is a codon corresponding to a site at which an amino acid is introduced.
[claim11]
11. A method of producing a protein comprising two different phosphor amino acids in an in-vitro translation system, wherein the method comprises preparing an mRNA into which a single 4-base codon and a single UAG codon have been inserted at specified positions; preparing two mutant tRNA molecules of claim 3 comprising anti-codons that pair with the 4-base codon and the UAG codon, and wherein one of the tRNA molecules is bound to an amino acid labeled with the fluorescent energy donor and the other tRNA is bound to an amino acid labeled with a fluorescent energy acceptor; and
allowing the translation system to synthesize the protein wherein, the protein encoded by said mRNA displays a change in distance and orientation upon interaction with other molecules and results in a change in the efficiency of fluorescence resonance energy transfer.
  • 発明者/出願人(英語)
  • HOHSAKA TAKAHIRO
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 536/23.1
  • 530/409
参考情報 (研究プロジェクト等) PRESTO Structure and Function of Biomolecules AREA
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