title: Energizing eukaryotic cell-free protein synthesis with glucose metabolism.
abstract: Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute E136, Evanston, IL USA, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL USA, 60208-3120.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Anderson
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute E136, Evanston, IL USA, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL USA, 60208-3120.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Stark
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute E136, Evanston, IL USA, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL USA, 60208-3120.
authors.forename: C Eric
authors.initials: CE
authors.lastname: Hodgman
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute E136, Evanston, IL USA, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL USA, 60208-3120.|Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 676 N. St Clair St, Suite 1200, Chicago, IL, USA, 60611-3068.|Simpson Querrey Institute, 303 E. Superior St, Suite 11-131 Chicago, IL USA, 60611-2875.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002474:Cell-Free System; D005947:Glucose / Q000378:metabolism*; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; D005947:Glucose
keywords: Cell-free biology; Cell-free protein synthesis; In vitro transcription and translation; Natural energy metabolism; Protein expression; Saccharomyces cerevisiae