Creation of functional peptides recognizing a low-molecular compound by evolutionary molecular engineering

通过进化分子工程创建识别低分子化合物的功能肽

• 批准号: 23510253
• 负责人: NEMOTO Naoto
• 金额: $ 3.41万
• 依托单位: Saitama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2013
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23510253/
• 关键词: 進化分子工学; ペプチド; アプタマー; アミノ基認識; 分子間相互作用; 試験管内淘汰; ｃDNA display; mRNA display; ペプチドアプタマー; 分子認識; 架橋構造; ジスルフィド結合; 進化工学; In vitro selection; cDNA display; Peptide aptamer; mRNA/cDNA display; 抗体低分子化; 低分子認識分子; 分子間相互作用解析; 表面プラズモン共鳴法; プルダウン法

We have succeeded to improve the preparation of cDNA display molecule easily and efficiently with a one-pot preparational manner. Furthermore, we demonstrated the biotin binding capacity of streptavidin magnetic beads decreased by mRNA-binding ribosomes, resulted in the low yield of cDNA display molecules. Secondly, we developed an easy and rapid molecular-protein interaction analysis using a cell-free translation and a novel puromycin-linker by a pull-down manner. Finally, by using these techniques, in vitro selection using 30 amino acids random library containing cysteine against amino group on the solid-phase was performed. As a result, an amino group-binding peptide aptamer containing two disulfide bonds which have a unique structure has explored. This shows that a peptide has capability to recognize small molecules with a low-molecular weight. In the future, peptides will be more and more applicable for nanotechnology as a recogniton material of nano sturcture.

我们成功地以一锅法制备方式，轻松高效地改进了cDNA展示分子的制备。此外，我们证明了链霉亲和素磁珠的生物素结合能力因mRNA结合核糖体而降低，导致cDNA展示分子的产量低。其次，我们通过下拉方式使用无细胞翻译和新型嘌呤霉素接头开发了一种简单快速的分子-蛋白质相互作用分析。最后，通过使用这些技术，使用针对固相上的氨基的含有半胱氨酸的30个氨基酸随机文库进行体外选择。结果，开发了一种具有独特结构的含有两个二硫键的氨基结合肽适体。这表明肽具有识别低分子量小分子的能力。未来，肽作为纳米结构的识别材料将越来越多地应用于纳米技术。

项目成果

A function of the primitive protein consisting of four kinds of amino acids

由四种氨基酸组成的原始蛋白质的功能

• 发表时间: 2012
• 作者: Shigefumi Kumachi; Miho Suzuki; Koichi Nishigaki; Yuzuru Husimi; Naoto Nemoto
• 通讯作者: Naoto Nemoto

Molecular Interactions Analysis by Fluorescence Correlation Spectroscopy(FCS) Method and Rapid Fluorescence Labeling of Proteins with a cell-free Translation System

荧光相关光谱 (FCS) 方法的分子相互作用分析以及无细胞翻译系统对蛋白质的快速荧光标记

• 发表时间: 2012
• 作者: Shigefumi Kumachi; Miho Suzuki; Koichi Nishigaki; Naoto Nemoto
• 通讯作者: Naoto Nemoto

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• 发表时间: 2012

Increasing the library size in cDNA display by optimizing purification procedures

通过优化纯化程序增加 cDNA 展示中的文库大小

• 发表时间: 2013
• 影响因子: 6.4
• 作者: Y. Mochizuki; S. Kumachi; K. Nishigaki; N. Nemoto
• 通讯作者: N. Nemoto