title: Evolution of translation initiation sequences using in vitro yeast ribosome display.
abstract: We report a novel in vitro yeast ribosome display method based on cell-free protein synthesis (CFPS) using linear DNA templates. We demonstrate that our platform can enrich a target gene from a model library by 100-fold per round of selection. We demonstrate the utility of our approach by evolving cap-independent translation initiation (CITI) sequences, which result in a 13-fold increase in CFPS yields after four rounds of selection, and a threefold further increase by placing the beneficial short sequences in tandem. We also show that 12 of the selected CITI sequences permit precise control of gene expression in vitro over a range of up to 80-fold by enhancing translation (and not as cryptic promoters). These 12 sequences are then shown to tune protein expression in vivo, though likely due to a different mechanism. Looking forward, yeast ribosome display holds promise for evolving libraries of proteins and DNA regulatory parts for protein engineering and synthetic biology. Biotechnol. Bioeng. 2016;113: 1777-1786. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.
authors.forename: Rui
authors.initials: R
authors.lastname: Gan
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208. m-jewett@northwestern.edu.|Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois. m-jewett@northwestern.edu.|Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois. m-jewett@northwestern.edu.|Simpson Querrey Institute, Northwestern University, Evanston, Illinois. m-jewett@northwestern.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D061505:Cell Surface Display Techniques; D002474:Cell-Free System; D019020:Directed Molecular Evolution / Q000379:methods*; D014176:Protein Biosynthesis / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D058615:Synthetic Biology
keywords: cap-independent translation initiation; directed evolution; ribosome display; synthetic biology; yeast cell-free protein synthesis