Protocol for preparation of cell–free system

Cell-free protein synthesis (CFPS) has emerged as a revolutionary process for producing proteins quickly and in a modular way, with the optionality of not having all of the restraints of living cells. As described in this chapter, we provide full protocols for preparing cell extracts with high activity, optimizing the reaction systems for protein expression, and establishing assays to characterize and evaluate protein expression and function. We included a detailed stepwise protocol for host strain selection, mechanical lysis, extract clarification, and optimization of energy and ionic balance. We evaluated several analytical techniques including SDS-PAGE and Western blot analysis and fluorescence assays as protocols for evaluating yield and functionality of the protein. For troubleshooting yield losses, we discussed some of the more common bottlenecks such as degradation of mRNA, protein misfolding, and contamination. CFPS offers the opportunity to expand applications for novel protein production and applications in synthetic biology, diagnostic fields, and on-demand biomanufacturing processes. CFPS can potentially produce proteins that are toxic, unstable, or non-natural. The future integrated process with combinations of machine learning and CFPS in freeze-dried formats are promising in that they will create scalable, mobile, and potentially deployable CFPS systems that can be utilized for food and healthcare applications as well as potential uses in industry. This work appears to be a suitable balancing act between serving as a protocol for synthesis of proteins, while also serving as a model and foresight for potential growth of a platform by researchers.