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

本研究开发了高效的无细胞合成各种cDNA蛋白质和亲和纯化的系统。为了提高无细胞蛋白质合成系统的效率，我们对两相系统和透析系统进行了比较。采用透析系统后，蛋白质的最终合成量显著提高。建立了分子伴侣和折叠酶的高效生产体系，并研究了它们对蛋白质折叠的影响。将伴侣蛋白与大肠杆菌二硫键异构酶（Dsb蛋白）结合，可显著提高蛋白质的复性效率。我们还开发了热响应性磁性纳米颗粒，用于固定化伴侣蛋白和折叠酶以及亲和分离。将N-丙烯酰甘氨酸酰胺（NAGAm）和生物素衍生物组成的温敏性聚合物包覆在平均粒径为10-20 nm的磁性纳米粒子上，制备了平均粒径为100 nm的温敏性磁性纳米粒子。这些磁性纳米粒子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。该温敏磁性纳米粒子可用于分子伴侣和折叠酶的固定化以及合成蛋白质的亲和分离。

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.