Chemical Expansion of the Central Dogma towards Synthetic Microorganisms

中心法则对合成微生物的化学扩展

• 批准号: 15101008
• 负责人: SISIDO Masahiko
• 金额: $ 66.31万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (S)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15101008/
• 关键词: Nonnatural amino acid; In vitro protein synthesis; Aminoacylation; tRNA; Nonnatural mutant protein; In vivo protein synthesis; Synthetic microorganism; 拡張翻訳系; 直交化tRNA; 翻訳伸張因子; 蛍光性アミノ酸; ペプチド核酸; 4塩基コドン

This research project aims to expand molecules like amino acids, nucleic acids and proteins constitute the protein biosynthesizing system to synthesize novel type of proteins with artificial functions. The final goal is to create synthetic microorganisms that live with 21 type of amino acids. During the 5 year we obtained the following results. (1) Some nonnatural amino acids were successfully charged onto a specific tRNA by the use of a peptide nucleic acid (PNA) that is complementary to the tRNA. The PNA-assisted aminoacylation mold be carried out in an E.coli in vitro protein biosynthesizing system to create a protein incorporated with the nonnatural amino add. (2) tRNAs and an elongation faint Tu. (EF-Tu) were expanded to bring large-sized nonnatural amino adds into E.col ribosome and subsequently to a protein. (3) An orthogonal set of aminoacyl tRNAsynthetase (ARS) and tRNA was screened to bring some nonnatural amino acids into proteins in living mammalian cells. (4) Finally these expanded molecular systems were introduced into mammalian cells and the synthesis of proteins containing nonnatural amino acids was detected, although its quantity was very small.In the last year, an attempt was made to apply these techniques to attach fluorescent amino acids to peptides and proteins. The fluorescently labeled peptides were screened to dimmer those that bind to cancer cells or to cancer-cell specific proteins.

本研究计划旨在扩展构成蛋白质生物合成系统的氨基酸、核酸和蛋白质等分子，以合成具有人工功能的新型蛋白质。最终的目标是创造出能与21种氨基酸共存的合成微生物。在这五年中，我们取得了以下成果。(1)通过使用与tRNA互补的肽核酸（PNA），一些非天然氨基酸被成功地加载到特定的tRNA上。在大肠杆菌体外蛋白质生物合成系统中进行PNA辅助的氨酰化，以产生掺入非天然氨基添加物的蛋白质。（EF-Tu）进行扩增，将大尺寸的非天然氨基添加到大肠杆菌核糖体中，随后添加到蛋白质中。(3)用氨酰tRNA合成酶（ARS）和tRNA的正交组合筛选出一组能将某些非天然氨基酸引入活的哺乳动物细胞蛋白质中的酶。(4)最后，这些扩展的分子系统被引入哺乳动物细胞，并检测到含有非天然氨基酸的蛋白质的合成，尽管其数量非常少。去年，人们试图应用这些技术将荧光氨基酸连接到肽和蛋白质上。筛选荧光标记的肽以使那些与癌细胞或癌细胞特异性蛋白质结合的肽变暗。

项目成果

T Transport of pyrrolidine-based oxy-peptide nucleic acids into c ytoplasm of CHO cells

T 将基于吡咯烷的氧肽核酸转运至 CHO 细胞的细胞质

• 发表时间: 2006
• 作者: M.;Kitamatsu;A.;Takahashi;R.;Matsuzaki;M.;Sisido
• 通讯作者: Sisido

"生体高分子"、「役にたつ化学シリーズ7.高分子化学」

《生物聚合物》、《实用化学系列7.高分子化学》

• 发表时间: 2005
• 作者: Ando;H;宍戸昌彦 他共著
• 通讯作者: 宍戸昌彦 他共著

目的タンパク質または目的ペプチドにアミノ

将氨基酸添加到您感兴趣的蛋白质或肽中。

• 发表时间: 2007

部位特異的アミノ酸導入法のための新規な直交化

位点特异性氨基酸引入方法的新颖正交化

• 发表时间: 2006

芳坂貴弘, 村中宣仁, 小宮山千絵, 松井絹枝, 高浦里美, 阿部亮二, 村上裕, 宍戸昌彦: "Position-Specific Incorporation of Dansylated Nonnatural Amino Acids into Strepotavidin by using a four-base codon"FEBS Letters. (印刷中). (2004)

Takahiro Yoshisaka、Nobuhito Muranaka、Chie Komiyama、Kinue Matsui、Satomi Takaura、Ryoji Abe、Yutaka Murakami、Masahiko Shishido：“使用四碱基密码子将丹酰化非天然氨基酸特定位置掺入链霉亲和素”FEBS Letters。 ）（2004）。