Automation and Screening DOI: 10.1177/2211068213501497

With the completion of the Human Genome Project in 2003 and the vast number of genes identified, understanding the structural and functional properties of the proteins encoded in these genes has become an important area of research in proteomics. Synthesizing proteins necessary for these stud-ies would typically involve cellular expression systems using primarily Escherichia coli.1–4 However, such cellular expression systems have several limitations, including the formation of inclusion bodies, protein degradation through proteolysis, lack of post-translational modification, and inability to synthesize cytotoxic proteins. Cellular expres-sion systems have been expanded to include eukaryotic cells to combat these limitations, but expression of cyto-toxic proteins and proteolysis are still issues.1–4 An alternative to protein expression in intact cells is cell-free protein synthesis (CFPS), which involves using the lysate from a cellular system for protein synthesis. CFPS requires the following components: a genetic template (e.g., DNA) with the target gene, the machinery necessary for transcription and translation (e.g., ribosomes), and a solu-tion containing energy molecules such as adenosine-5′-triphosphate (ATP) and nutrients (e.g., amino acids) necessary to sustain protein expression. Compared with traditional pro-tein expression within an intact cell, CFPS reduces protein synthesis time, eliminates cytotoxicity concerns, reduces proteolysis, and allows greater control over the chemicals included for protein synthesis.5,6 These advantages have enabled CFPS applications to be expanded to structural and functional proteomics,7–9 synthesis and characterization of cytotoxic proteins,10,11 drug discovery and screening,12,13 and high-throughput protein synthesis.14–16 CFPS has been performed in three formats. The first one is a simple batch format characterized by low protein syn-thesis yields and long processing times as a result of th