Cell-Free PURE System: Evolution and Achievements

Abstract

The cell-free protein synthesis (CFPS) system, as a technical core of synthetic biology, can simulate the transcription and translation process in an in vitro open environment without a complete living cell. It has been widely used in basic and applied research fields because of its advanced engineering features in flexibility and controllability. Compared to a typical crude extract-based CFPS system, due to defined and customizable components and lacking protein-degrading enzymes, the protein synthesis using recombinant elements (PURE) system draws great attention. This review first discusses the elemental composition of the PURE system. Then, the design and preparation of functional proteins for the PURE system, especially the critical ribosome, were examined. Furthermore, we trace the evolving development of the PURE system in versatile areas, including prototyping, synthesis of unnatural proteins, peptides and complex proteins, and biosensors. Finally, as a state-of-the-art engineering strategy, this review analyzes the opportunities and challenges faced by the PURE system in future scientific research and diverse applications.

Cell-free protein synthesis for nonribosomal peptide synthetic biology

“…The crude cell lysates are generated from cell growth and then are supplemented with cofactors and other necessary biological components for in vitro transcription and translation (TX-TL). Whereas TX-TL PURE system or PURExpress (Shimizu et al, 2001;Tuckey et al, 2014;Lavickova and Maerkl, 2019;Cui et al, 2022) is a system that has taken the minimal recombinant elements required for cell-free expression and is thus less complex than lysate-based systems. The lysate-based system has the advantage over the PURExpress system in that it allows for accessory genes (e.g., ones relevant to posttranslational modification) as well as chaperonins.…”