title: Improved cell-free RNA and protein synthesis system.
abstract: Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Liangcai
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Aach
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002474:Cell-Free System; D006160:Guanosine Triphosphate / Q000378:metabolism; D049409:Luciferases, Firefly / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis*; D012343:RNA, Transfer / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D046911:Macromolecular Substances; D018832:Molecular Chaperones; D010445:Peptide Elongation Factors; D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D049409:Luciferases, Firefly