| Summary: | With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for <i>Escherichia coli</i>-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered <i>E. coli</i> strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for <i>E. coli</i> cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active <i>E. coli</i> cell extract.
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