A Method for High-Throughput Screening of Initial Codons Engineered for Maximal Production of Recombinant Proteins

Cell-free protein synthesis system provides an open environment for gene expression and allows direct access to the molecular events of transcription and translation. In this study, we demonstrate the use of cell-free protein synthesis techniques for determining the initial codons that can lead to maximized production of target proteins. We first randomized the first two codons, and the individual variant genes were co-expressed with the gene of a reporter protein (sfGFP) in the same mixture. As a results of the competition between the variant gene and a common reporter gene for the finite pool of ribosome in the reaction mixture, we could readily estimate the relative expression levels of the variant genes by inverse correlation between the sfGFP fluorescence and the expression levels of target genes [3]. Through this simple method, we could rapidly screen for the initial codons that substantially enhances the production of a target protein. Importantly, the initial codons identified by the cell-free co-expression method also effectively enhanced protein production in E. coli, indicating that this method can be used as a general tool to predict and modulate the yield of protein production during cell-based gene expression.