Site-selective introduction of unnatural amino acid into proteins and its use for analyses of structure-function relationship of proteins.
| Project/Area Number |
16310148
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | Gifu University |
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Principal Investigator |
NISHIKAWA Kazuya Gifu University, Faculty of Engineering, Professor, 工学部, 教授 (60109262)
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| Co-Investigator(Kenkyū-buntansha) |
YOKOGAWA Takashi Gifu University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (90242304)
OHNO Satoshi Gifu University, Faculty of Engineering, Research Associate, 工学部, 助手 (10345796)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2006: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Keywords | Biotechnology / Protein / Unnatural Amino Acid / Cell-free Protein-synthesis / Bio-molecule / 雌 / アジドチロシン / サプレッサーtRNA / ブロモチロシン |
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| Research Abstract |
1. E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA^<Tyr> (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA^<Tyr>/ tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering.
2. Yeast mitochondrial tRNA^<Tyr> was shown to be possibly used as an Opal-suppressor and a mutant (F38A) of yeast mitochondrial tryptophanyl-tRNA synthetase having an altered amino acid specificity was created by genetic engineering.
3. An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azido-tyrosine has been developed. Using rat calmodulin as a model protein, we prepared several unnatural calmodulin molecules, each carrying an azido-tyrosine at predetermined positions. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated calmodulin molecules. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g. probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized.
4. As an important supporting technique for X-ray crystallographic analyses (SAD method), we have developed an effective method for incorporating 3-iodo-tyrosine into site-selective positions in a target protein. We have also determined the crystal structure of yeast TyrRS complexed with a Tyr-AMP analogue and the native tRNA^<Tyr> at 2.4 Å resolution.
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Report
(4 results)
Research Products
(31 results)
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[Journal Article] Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.2007
Author(s)
M.Abe, S.Ohno, T.Yokogawa, T.Nakanishi, F.Arisaka, T.Hosoya, T.Hiramatsu, M.Suzuki, T.Ogasawara, T.Sawasaki, K.Nishikawa, M.Kitamura, H.Hori, Y.Endo
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Journal Title
Proteins 67(3)
Pages: 643-652
Description
「研究成果報告書概要(和文)」より
Related Report
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[Journal Article] Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.2007
Author(s)
M.Tsunoda, Y.Kusakabe, N.Tanaka, S.i Ohno, M.Nakamura, T.Senda, T.Moriguchi, N.Asai, M.Sekine, T.Yokogawa, K.Nishikawa, K.T.Nakamura
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Journal Title
Nucleic Acids Res. (in press)
Description
「研究成果報告書概要(和文)」より
Related Report
-
[Journal Article] Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.2007
Author(s)
M.Abe, S.Ohno, T.Yokogawa, T.Nakanishi, F.Arisaka, T.Hosoya, T.Hiramatsu, M.Suzuki, T.Ogasawara, T.Sawasaki, K.Nishikawa, M.Kitamura, H.Hori, Y.Endo
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Journal Title
Proteins 67 (3)
Pages: 643-652
Description
「研究成果報告書概要(欧文)」より
Related Report
-
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[Journal Article] Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.2007
Author(s)
M.Tsunoda, Y.Kusakabe, N.Tanaka, S.Ohno, M.Nakamura, T.Senda, T.Moriguchi, N.Asai, M.Sekine, T.Yokogawa, K.Nishikawa, K.T.Nakamura
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Journal Title
Nucleic Acids Res. (in press)
Description
「研究成果報告書概要(欧文)」より
Related Report
-
[Journal Article] Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.2007
Author(s)
M.Abe, S.Ohno, T.Yokogawa, T.Nakanishi, F.Arisaka, T.Hosoya, T.Hiramatsu, M.Suzuki, T.Ogasawara, T.Sawasaki, K.Nishikawa, M.Kitamura, H.Hori, Y.Endo
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Journal Title
proteins 67(3)
Pages: 643-652
Related Report
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[Journal Article] Crystallization and preliminary X-ray crystallographic analysis of yeast tyrosyl-tRNA synthetase complex with its cognate tRNA.2006
Author(s)
Y.Kusakabe, S.Ohno, N.Tanaka, M.Nakamura, M.Tsunoda, T.Moriguchi, N.Asai, M.Sekine, T.Yokogawa, K.Nishikawa, K.T.Nakamura
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Journal Title
Protein and Peptide Letters 13(4)
Pages: 417-419
Description
「研究成果報告書概要(和文)」より
Related Report
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[Journal Article] Crystallization and preliminary X-ray crystallographic analysis of yeast tyrosyl-tRNA synthetase complex with its cognate tRNA.2006
Author(s)
Y.Kusakabe, S.Ohno, N.Tanaka, M.Nakamura, M.Tsunoda, T.Moriguchi, N.Asai, M.Sekine, T.Yokogawa, K.Nishikawa, K.Nakamura
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Journal Title
Protein and Peptide Letters 13 (4)
Pages: 417-419
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Crystallization and preliminary X-ray crystallographic analysis of yeast tyrosyl-tRNA synthetase complex with its cognate tRNA2006
Author(s)
Kusakabe, S.Ohno, N.Tanaka, M.Nakamura, M.Tsunoda, T.Moriguchi, N.Asai, M.Sekine, T.Yokogawa, K.Nishikawa, K, Nakamura.
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Journal Title
Protein and Peptide Letters 13,4
Pages: 417-419
Related Report
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