Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract.

Cell-free protein synthesis (CFPS) has become an established biotechnology tool for rapid protein expression. Over the past few decades many advances have elevated CFPS from a niche, low-efficiency system to one now capable of biomanufacturing custom proteins. Many research papers and reviews exist on the advances made in CFPS genetic template and cell extract preparation for use in E. coli systems. What is currently missing from the literature is a comprehensive review on the myriad of supplement recipes added to the CFPS reaction to support metabolism, transcription, and translation. This list of supplements has changed over the years, with a general drive towards greater simplification. Herein we provide a comprehensive list of the supplements used in CFPS, tracing major recipe classes as the field has evolved. We also provide an in-depth analysis of the proposed biochemical purpose each supplement has in the reaction. This review reveals the significance of correct supplements on overall CFPS productivity; however, the large range of supplements accommodated by CFPS also shows an inherent flexibility in the CFPS reaction as well as additional room for optimization and recipe simplification.