Development of high efficiency cell-free protein production and purification system

高效无细胞蛋白生产和纯化系统的开发

• 批准号: 13650830
• 负责人: KONDO Akihiko
• 金额: $ 2.62万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650830/
• 关键词: mlecular chaperon; foldase; fine particles; cell-free protein synthesys; stimuli-sensitive polymer; protein; folding; affinity separation; 磁性微粒子; 温度応答性微粒子; 高度分離

In this study, efficient system for cell-free synthesis of proteins from various cDNA and affinity purification was developed. To improve the efficiency of cell-free protein synthesis system, we compared the two phase system and dialysis system. By adopting dialysis system, the final amount of synthesized protein increased significantly. Then, we established the efficient production system of molecular chaperons and foldases, and their effects on the protein folding were investigated. By combining chaperonin and disulfide isomerase (Dsb protein) from Escherichia coli, protein refolding efficiency was improved significantly. We have also developed thermo-responsive magnetic nanoparticles for immobilization of chaperonin and foldases and affinity separation. The thermno-responsive magnetic nanoparticles (average diameter of 100 nm) were synthesized by coating magnetite nanoparticles (average diameter of 10-20 nm) with a thermoresponsive polymer consisting of N-acryloilglycine amide (NAGAm) and biotin derivative. These magnetic nanoparticles showed a reversible transition between flocculation and dispersion at around upper critical flocculation temperature. The thermo-responsive magnetic nanoparticles were proved to be effective for immobilization of molecular chaperones and foldases and affinity separation of synthesized proteins.

在这项研究中，开发了各种cDNA和亲和力纯化的蛋白质无细胞合成的有效系统。为了提高无细胞蛋白质合成系统的效率，我们比较了两个相系统和透析系统。通过采用透析系统，合成蛋白的最终量显着增加。然后，我们建立了分子伴侣和折叠酶的有效生产系统，并研究了它们对蛋白质折叠的影响。通过将大肠杆菌的伴侣蛋白和二硫化物异构酶（DSB蛋白）结合在一起，可显着提高蛋白质的再折叠效率。我们还开发了热响应性磁性纳米颗粒，用于固定伴侣蛋白和折叠酶以及亲和力分离。通过涂层磁铁矿纳米颗粒（平均直径为10-20 nm）与由N-乙烯丙烯酰基甘氨酸酰胺酰胺（Nagam）和生物素衍生物合成，通过涂层磁铁矿纳米颗粒（平均直径为10-20 nm）合成热反应性磁性纳米颗粒（平均直径为100 nm）。这些磁性纳米颗粒显示在临界絮凝温度上的絮凝和分散体之间存在可逆的过渡。事实证明，热响应的磁性纳米颗粒有效地固定分子伴侣和折叠酶以及合成蛋白的亲和力分离。

项目成果

Hrotaka Furukawa: "Affinity selection of target cells from cell surface displayed libraries : A nobel procedure using thermo-responsive magnetic nanoparticles"Applied Microbiology and Biotechnology. (in press).

Hrotaka Furukawa：“从细胞表面展示的文库中亲和选择靶细胞：使用热响应磁性纳米颗粒的诺贝尔程序”应用微生物学和生物技术。

Hirotaka Furukawa: "Affinity selection of target cells from cell surface displayed libraries : A nobel procedure using thermo-responsive magnetic nanoparticles"Applied Microbiology and Biotechnology. in press.

Hirotaka Furukawa：“从细胞表面展示的文库中亲和选择靶细胞：使用热响应磁性纳米颗粒的诺贝尔程序”应用微生物学和生物技术。

Akihiko Kondo: "Development of magnetic nanoparticles and their application to bionano-technology"Expected Materials for the Future. 2(10). 19-25 (2002)

近藤明彦：“磁性纳米粒子的开发及其在生物纳米技术中的应用”未来的预期材料。

Jiro Kohda: "Improvement of productivity of active form of glutamate racemase in escherichia coli by coexpression of folding accessory proteins"Biochemical Engineering Journal. 10(1). 39-45 (2002)

Jiro Kohda：“通过折叠辅助蛋白的共表达提高大肠杆菌中谷氨酸消旋酶活性形式的生产力”生物化学工程杂志。

Jiro Kohda: "Refolding of proteins by hexadecamers and monomers of and subunits of group II chaperonin from hyperthermophilic archaeum, thermococcus strain KS-1"Biochemical Engineering Journal. in press.

Jiro Kohda：“来自超嗜热古细菌、热球菌菌株 KS-1 的十六聚体和 II 组伴侣蛋白单体和亚基对蛋白质的重折叠”《生物化学工程杂志》。