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 and translation 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 were assessed, including 5"-UTR, 3"-UTR, and length of poly(A) tail. A specific vector harboring the 5"-UTR fragment of... More
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 and translation 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 were 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 length 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 transcription and translation 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-hour 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 and holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site (IRES), and production of protein libraries for genome scale studies.