title: Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis.
abstract: Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1)  µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA.
authors.forename: C Eric
authors.initials: CE
authors.lastname: Hodgman
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002457:Cell Extracts; D002474:Cell-Free System / Q000382:microbiology*; D005285:Fermentation; D049409:Luciferases, Firefly / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D011994:Recombinant Proteins; D049409:Luciferases, Firefly
keywords: Saccharomyces cerevisiae; cell-free biology; cell-free protein synthesis; in vitro translation; protein expression