Development of Signal Peptide Optimization Tool

信号肽优化工具的开发

• 批准号: 23656522
• 负责人: NAKANO Hideo
• 金额: $ 2.5万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-23656522/
• 关键词: 生物機能工学; 微生物; 分泌生産; シグナルペプチド; 酵素; 酵母; ゲノムライブラリー

The secretion level of foreign protein in recombinant microbe is strongly dependent onthe combination of signal peptides (SPs) and the target protein; therefore optimization of SP sequence to each target protein is a crucial step to maximize the productivity of the secreted protein in heterogenous expression both in prokaryote and eukaryote.In this research, we have developed a novel method to adjust SP for each target protein utilizing the library of endogenous signal peptides of host cells, named signal peptide optimization tool (SPOT), which can easily make a library of SP fused to a target protein without any interfacial sequence. As a model system, beta-galactosidase from Aspergillus oryzae was used as a target protein for the secretion from Saccharomyces secrevisiae, a typical eukaryotic expression host, and demonstrated that several SPs giving a larger amount of secreted protein from the recombinant yeast were found by the screening of relatively a small number of independent clones. In addition, a correlation between the secretion efficiency and the D-score of each signal peptide analyzed by Signal P software was found, suggesting the use of the software would be helpful to increase the quality of the library. The developed method can be used for variety of proteins in a variety of host cells.

外源蛋白在重组菌中的分泌水平强烈依赖于信号肽与目的蛋白的结合;因此，针对每个靶蛋白的SP序列的优化是在原核生物和真核生物的异源表达中使分泌蛋白的产量最大化的关键步骤。在本研究中，我们开发了一种利用宿主细胞内源性信号肽库来调整每个靶蛋白SP的新方法，称为信号肽优化工具（SPOT），其可以容易地制备与靶蛋白融合的SP文库而无需任何界面序列。作为一个模型系统，从曲霉β-半乳糖苷酶被用作从分泌酵母，一个典型的真核表达宿主分泌的靶蛋白，并证明了几个SP给出大量的分泌蛋白的重组酵母被发现的筛选相对较少的独立克隆。此外，通过Signal P软件分析，发现每个信号肽的分泌效率与D值之间存在相关性，表明该软件的使用将有助于提高文库的质量。开发的方法可用于多种宿主细胞中的多种蛋白质。

项目成果

酵母シグナルペプチドライブラリーを用いたシグナルペプチド最適化ツールの開発に関する研究

利用酵母信号肽库开发信号肽优化工具的研究

• 发表时间: 2012
• 作者: 原翔一;菅原知宏;佐原健彦;扇谷悟;河原崎泰昌;兒島孝明;中野秀雄
• 通讯作者: 中野秀雄

酵母シグナルペプチドライブラリーを用いたシグナルペプチド最適化ツールの開発

利用酵母信号肽库开发信号肽优化工具

• 发表时间: 2012
• 作者: 原翔一;ら
• 通讯作者: ら