Cell-free protein synthesis is a promising technique for the rapid production of proteins. However, the application of the cell-free systems requires the development of an artificial chaperone that prevents aggregation of the protein and supports its correct folding. Here, nanogel-based artificial chaperones are introduced that improve the folding efficiency of rhodanese produced in cell-free systems. Although rhodanese suffers from rapid aggregation, rhodanese was successfully expressed in the presence of the nanogel and folded to the enzymatically active form after addition of cyclodextrin. To validate the general applicability, the cell-free synthesis of ten water-soluble proteins was examined. It is concluded that the nanogel enables efficient expression of proteins with strong aggregation tendency.
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