Reconstituted cell-free translation systems for exploring protein folding and aggregation

Protein folding is crucial for achieving functional three-dimensional structures. However, the process is often hampered by aggregate formation, necessitating the presence of chaperones and quality control systems within the cell to maintain protein homeostasis. Despite a long history of folding studies involving the denaturation and subsequent refolding of translation-completed purified proteins, numerous facets of cotranslational folding, wherein nascent polypeptides are synthesized by ribosomes and folded during translation, remain unexplored. Cell-free protein synthesis (CFPS) systems are invaluable tools for studying cotranslational folding, offering a platform not only for elucidating mechanisms but also for large-scale analyses to identify aggregation-prone proteins. This review provides an overview of the extensive use of CFPS in folding studies to date. In particular, we discuss a comprehensive aggregation formation assay of thousands of Escherichia coli proteins conducted under chaperone-free conditions using a reconstituted translation system, along with its derived studies.