title: A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthe.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Gan
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004247:DNA
keywords: Cell-free protein synthesis; Combined transcription-translation; In vitro; Yeast