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.

在本研究中，开发了从各种 cDNA 中无细胞合成蛋白质和亲和纯化的有效系统。为了提高无细胞蛋白质合成系统的效率，我们比较了两相系统和透析系统。采用透析系统，最终合成蛋白质的量显着增加。然后，我们建立了分子伴侣和折叠酶的高效生产系统，并研究了它们对蛋白质折叠的影响。通过结合伴侣蛋白和大肠杆菌的二硫键异构酶（Dsb蛋白），蛋白质重折叠效率显着提高。我们还开发了热响应磁性纳米粒子，用于固定伴侣蛋白和折叠酶以及亲和分离。通过用由N-丙烯酰甘氨酰胺(NAGAm)和生物素衍生物组成的热响应聚合物涂覆磁铁矿纳米粒子(平均直径10-20 nm)合成热响应磁性纳米粒子(平均直径100 nm)。这些磁性纳米颗粒在上临界絮凝温度附近表现出絮凝和分散之间的可逆转变。热响应磁性纳米粒子被证明可有效固定分子伴侣和折叠酶以及合成蛋白质的亲和分离。

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》。