A combined cell‐free transcription‐translation system from Saccharomyces cerevisiae for rapid and robust protein synthe

Cell‐free protein synthesis (CFPS) provides a valuable platform for understanding, using, and expanding the capabilities of the translation apparatus. For example, high‐throughput CFPS is helping to address the increasing discrepancy between genome sequence data and their translation products. Here, we report the development of a combined cell‐free transcription‐translation (Tx/Tl) system from Saccharomyces cerevisiae that is suitable for such efforts. First, we show the ability to enable translation initiation in a cap‐independent manner. The performance of various genetic elements was assessed, including 5'‐UTR, 3'‐UTR, and length of poly(A) tail. A specific vector harboring the 5'‐UTR fragment of the Ω sequence from the tobacco mosaic virus and a poly(A) tail of 50 nucleotides led to optimal performance. Second, we developed a simple, two‐step polymerase chain reaction (PCR) method for high‐throughput production of linear templates for yeast CFPS. This procedure allows all functional elements needed for Tx/Tl to be added to an open‐reading frame directly by overlap extension PCR. Our two‐step PCR method was successfully applied to three reporter proteins: luciferase, green fluorescence protein, and chloramphenicol acetyl transferase, yielding 7 to 12.5 μg mL–1 active protein after 1.5‐h batch reactions. Surprisingly, the linear templates outperformed plasmid DNA by up to 60%. Hence, the presented CFPS method has the potential to rapidly prepare tens to thousands of DNA templates without time‐consuming cloning work. Further, it holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site, and production of protein libraries for genome‐scale studies. See accompanying commentary by Russ and Dueber DOI: 10.1002/biot.201400071