Construction of highly condensed protein/peptide library on micro-reactor array

微反应器阵列上高浓缩蛋白/肽库的构建

• 批准号: 12450332
• 负责人: NAKANO Hideo
• 金额: $ 9.41万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12450332/
• 关键词: cell-free protein synthesis; protein library; single-molecule PCR; micro-chamber array; lipase; GFP; high-throughput screening; antibody; ライブラリー; ビーズ; 均一性; 蛍光特性; マイクロアレイ; 蛍光蛋白質; PCR; 一本鎖抗体; ハイスループット

Herein we have developed a novel protein library system termed SIMPLEX : single-molecule PCR linked in vitro expression combining PCR amplification of single DNA molecule diluted in a well and cell-free protein synthesis. Use of single primer amplification and hot-startable DNA polymerase enabled a single step single-molecule PCR. The obtained protein library by SIMPLEX has been demonstrated to be highly uniform. The uniformity was actually much higher than that obtained by a conventional in vivo system. Also multi-molecule PCR in a well has been shown to be possible. These results have demonstrated that SIMPLEX can provide a high-throughput method for the construction and screening of protein library. Moreover, protein could be synthesized on a micro-chamber array with the concentration of 10,000/cm^2, which will lead the construction of highly condensed protein library.In addition, in vitro combinatorial mutagenesis was also developed to demonstrate that combinatorial mutation in a specific region of protein selected based on its 3D structure is effective to modulate or improve some properties of proteins.

在此，我们开发了一种新的蛋白质文库系统称为SIMPLEX：单分子PCR连接的体外表达结合PCR扩增的单个DNA分子在一个孔中稀释和无细胞蛋白质合成。单引物扩增和热启动DNA聚合酶的使用使得能够实现单步单分子PCR。通过SIMPLEX获得的蛋白质文库具有高度的均一性。均匀性实际上比通过常规体内系统获得的均匀性高得多。此外，已经证明孔中的多分子PCR是可能的。这些结果表明，SIMPLEX可以为蛋白质文库的构建和筛选提供一种高通量的方法。此外，我们还开展了体外组合突变实验，证明了基于蛋白质三维结构选择的特定区域的组合突变可以有效地调节或改善蛋白质的某些性质。