Cell-free protein synthesis energized by slowly-metabolized maltodextrin

Abstract

Background: Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS.

Results: Distinct from common secondary energy sources (e.g., phosphoenolpyruvate - PEP, glucose-6-phosphate), maltodextrin was used for energizing CFPS through substrate phosphorylation and the glycolytic pathway because (i) maltodextrin can be slowly catabolized by maltodextrin phosphorylase for continuous ATP regeneration, (ii) maltodextrin phosphorylation can recycle one phosphate per reaction for glucose-1-phosphate generation, and (iii) the maltodextrin chain-shortening reaction can produce one ATP per glucose equivalent more than glucose can. Three model proteins, esterase 2 from Alicyclobacillus acidocaldarius, green fluorescent protein, and xylose reductase from Neurospora crassa were synthesized for demonstration.

Conclusion: Slowly-metabolized maltodextrin as a low-cost secondary energy compound for CFPS produced higher levels of proteins than PEP, glucose, and glucose-6-phospahte. The enhancement of protein synthesis was largely attributed to better-controlled phosphate levels (recycling of inorganic phosphate) and a more homeostatic reaction environment.

Figure 1

Pathway of maltodextrin phosphorolysis and glycolysis for regeneration of ATP and recycling of inorganic phosphate. Maltodextrin phosphorylase, MP; phosphoglucomutase, PGM; and hexokinase, HK.

Figure 2

The synthesized EST2 energized by maltodextrin (MD), glucose, G6P, and PEP. (A) Profile of the EST2 synthesis levels based on its activity assays. The data were measured in triplicate. (B) Images of SDS-PAGE with stained esterase activity (magenta bands) for cell-free protein synthesis after incubation for 3 hours.

Figure 3

Profile of inorganic phosphate, pH and ATP levels. (A) inorganic phosphate concentration, (B) pH, and (C) ATP levels in CFPS energized by maltodextrin, glucose, G6P, and PEP, respectively. The data were measured in triplicate.

Figure 4

The CFPS for active EST2, active CBM-GFP, and purified xylose reductase by using different secondary energy compounds. The data were measured in triplicate.