Rapid Screening of Molecular Chaperones By Sequential Cell-Free Protein Synthesis Reactions

Functional expression of recombinant protein is a constant issue in diverse fields of biotechnology. In this study, taking advantage of the configurational flexibility of cell-free protein synthesis, we developed a method for rapid screening of optimal molecular chaperones that facilitate functional expression of target proteins. After the synthesis of a series of molecular chaperones, the reaction mixture for cell-free protein synthesis was reprogrammed with the genes of target proteins. The effect of a pre-synthesized molecular chaperone on protein folding was determined by measuring the enzymatic activity of target proteins synthesized in the second reactions. When tested for a number of recombinant enzymes that otherwise form insoluble aggregates, this method could successfully identify the molecular chaperones and their combinations that substantially improved the soluble and functional expression of those enzymes. 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.