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.

Keywords: Cell-free biology; Cell-free protein synthesis; In vitro transcription and translation; Natural energy metabolism; Protein expression; Saccharomyces cerevisiae.

Figure 1. Glycolysis is active in yeast crude extract CFPS

(A) Schematic of creatine phosphate (CrP)/creatine kinase (CrK) energy regeneration system. (B) Proposed glycolytic energy regeneration system in yeast crude extracts. (C) To assess the possibility of using glycolytic intermediates to fuel CFPS, six glycolytic intermediates (fructose 1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), glucose, 3-phosphglyceric acid (3-PGA), pyruvate, and glucose 6-phosphate (G6P)) were added as the sole secondary energy substrate to different yeast CFPS reactions in concentrations ranging from 0 mM to 30 mM and compared to a control composed of no secondary energy substrate (circle). Of the non-phosphorylated secondary energy substrates assessed, glucose is the highest yielding for yeast CFPS. (D) Time course reactions of active luciferase for several glycolytic intermediates for equivalent of 30 mM total carbon (e.g., 5 mM glucose or 10 mM PEP) and (E) HPLC analysis of ethanol production after 4-hour incubation for reactions performed in panel D. The numbers above each column denote the percentage of theoretical conversion of each secondary energy substrate to ethanol. Values shown are means with error bars representing the standard deviation of at least three independent experiments.

Figure 2. Yeast CFPS CrP/CrK plus glucose dual system for energy regeneration does not improve CFPS yields

(A) 0 to 25 mM glucose was added to CFPS reactions containing 25 mM creatine phosphate (CrP) and 0.27 mg/mL creatine kinase (CrK). Increasing the starting glucose concentration decreases luciferase yields. (B) The pH of CFPS reactions containing 25 mM CrP, 0.27 mg/mL CrK, and either 0 mM or 25 mM glucose was measured at regular intervals. (C) To assess possible ethanol inhibition, various concentrations of ethanol, ranging from 0 mM to 25 mM, were added to CFPS reactions. Active luciferase yields are reported relative to the 0 mM ethanol condition, showing that inhibition was not observed. (D) The concentration of ATP was measured at intervals during CFPS reactions including 25 mM CrP, 0.27 mg/mL CrK, and 0 to 25 mM glucose. ATP is rapidly depleted as the starting glucose concentration is increased. Values shown in A-C are means with error bars representing the standard deviation of at least three independent experiments. Data from panel D traces are individual measurements.

Figure 3. Optimizing yeast CFPS reaction conditions with glucose as a secondary energy substrate

(A) The optimal starting concentration of glucose was determined via addition of 0-30 mM of glucose to CFPS reactions containing 0.15 mM cAMP. The optimum was observed at 16 mM glucose. (B) Luciferase and (C) ATP concentrations were measured at regular intervals over time in CFPS reactions containing 16 mM glucose or 0 mM glucose. Values shown are means with error bars representing the standard deviation of at least three independent experiments.

Figure 4. CFPS reactions with glucose are phosphate-limited: Increasing phosphate concentration increases protein yields and prolongs the CFPS reaction

(A) The optimal amount of exogenous phosphate was determined via addition of 0-50 mM of phosphate to CFPS reactions containing 16 mM glucose. (B) Luciferase and (C) ATP concentration were measured at regular intervals in CFPS reactions containing 16 mM glucose and 25 mM phosphate or 0 mM glucose + 0 mM phosphate. Values shown are means with error bars representing the standard deviation of at least three independent experiments.