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

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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.