Rapid determination of effective folding agents by sequential cell-free protein synthesis

Cell-free protein synthesis enables the direct translation of genetic information into the corresponding proteins, and thus provides a powerful platform for functional assays of protein-coding genes. In this study, taking advantage of the configurational flexibility of cell-free protein synthesis, we developed a method to study the effect of molecular chaperones on the functional synthesis of recombinant proteins. After the synthesis of a series of molecular chaperones, the reaction mixture for cell-free protein synthesis was reprogrammed for synthesis of Candida antarctica lipase B (CalB). The effect of a pre-synthesized molecular chaperone on protein folding was determined by measuring the enzymatic activity of CalB synthesized in the second reaction. Using this sequential expression experiment, we could rapidly determine the molecular chaperones that effectively assisted the functional synthesis of CalB. We believe that the presented strategy will provide a versatile platform for the optimal production of functional proteins, and can also be extended to studies of other interacting proteins.