Integration of Non-natural Amino Acids Into Protein

将非天然氨基酸整合到蛋白质中

• 批准号: 04453161
• 负责人: NISHIKAWA Kazuya
• 金额: $ 3.97万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04453161/
• 关键词: Biosynthesis of proteins; Non-natural amino acids; Microsurgery of RNA; Artificial proteins

1) Several protecting groups of 5'-terminal and internucleotide phosphates and base amines were tested to obtain better yield of aminoacyl pCpCpA.Biotinyl-lysyl pCpA was designed and synthesized to make it easier to detect the amino acid after integration into proteins.2) An efficient method for preparing tRNA which has an anticodon of arbitrary nucleotide sequence and lacks 3'-terminal pCpCpA,was developed by the use of mictosurgery technique and yeast tRNA^<Phe>, tRNA^<Tyr> and so on. Furthermore, a non-radioactive method for determining nucleotide sequences of low-molecular-weight RNA was developed.3) Advantage in nuclease resistance and translational efficiency of phosphorothioate-containing mRNA (thio-mRNA) over natural mRNA was demonstrated in E.coli cell-free translation system. Effects of unusual polyamines on phenylalanyl-tRNA formation was investigated in cell-free systems of an extreme thermophile, Thermus themophilus HB8. Fundamental characteristics of cell-tree translation systems from yeast, Candida cylindracea and bovine mitochondriawere also examined.

1)为了获得更好的氨基酰基pCpCpA产率，对5′端和核苷酸间磷酸基和碱基胺的多个保护基团进行了测试。设计并合成了生物素基赖氨酸聚羧酸酯，使氨基酸整合到蛋白质后更容易检测。2)利用显微外科技术和酵母tRNA^<Phe>、tRNA^<Tyr>等条件，开发了一种制备具有任意核苷酸序列反密码子且缺乏3'端pCpCpA的tRNA的有效方法。此外，还开发了一种测定低分子量RNA核苷酸序列的非放射性方法。3)在大肠杆菌无细胞翻译系统中证实了含硫代磷酸酯mRNA （thio-mRNA）在抗核酸酶和翻译效率方面优于天然mRNA。在极端嗜热菌HB8的无细胞系统中，研究了不寻常的多胺对苯丙酰trna形成的影响。细胞树翻译系统的基本特征，从酵母，念珠菌和牛线粒体也进行了检查。

项目成果

T.Uzawa,A.Yamagishi,K.Nishikawa and T.Oshima: "Effects of unusual polyamines on phenylalanyl-tRNA formation." Journal of Biochemistry. 115. 830-832 (1994)

T.Uzawa、A.Yamagishi、K.Nishikawa 和 T.Oshima：“异常多胺对苯丙氨酰-tRNA 形成的影响。”

Tatsuya Niimi: "Recognition of the Auticodon Loop of tRNA^<Ile>_1 by Isoleucyl-tRNA Synthetase from Escherichia coli" Nucleosides and Nucleotides. (印刷中). (1994)

Tatsuya Niimi：“大肠杆菌异亮氨酰-tRNA 合成酶对 tRNA^<Ile>_1 的自密码子环的识别”核苷和核苷酸（1994 年出版）。

K.Ogawa, S.Takemura, and K.Nishikawa: "Anticodon Loop Structure of Torulopsis utilis tRNA^<Val> and Valine Acceptance." J.Adv.Sci.5. 43-49 (1993)

K.Okawa、S.Takemura 和 K.Nishikawa：“Torulopsis utilis tRNA^<Val> 的反密码子环结构和缬氨酸接受。”

Takahiro Matsuki: "Sequence Analysis of low-molecular-weight RNAs by the use of non-radioactive labeling" Nucleic Acids Symp.Ser.29. 75-76 (1993)

Takahiro Matsuki：“使用非放射性标记对低分子量 RNA 进行序列分析”核酸 Symp.Ser.29。

Norihiko Yamazaki: "Purification and characterization of tRNA(adenosine-1)-methyltransferase from Thermus thermophilus HB27" Nudeic Acids Symp.Ser.27. 141-142 (1922)

Norihiko Yamazaki：“来自嗜热栖热菌 HB27 的 tRNA（腺苷-1）-甲基转移酶的纯化和表征”Nudeic Acids Symp.Ser.27。