Cell-free protein synthesis energized by slowly-metabolized maltodextrin

Yiran Wang,Y-H Percival Zhang

doi:10.1186/1472-6750-9-58

Abstract

BackgroundCell-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.ResultsDistinct 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.ConclusionSlowly-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.

Highlights

Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes
Maltodextrin was slowly cleaved into glucose-1-phosphate (G1P) in the presence of inorganic phosphate catalyzed by maltodextrin phosphorylase (Eq 1)
We tested the effects of maltodextrin phosphorylase and phosphoglucomutase loading on the EST2 yields

Summary

Introduction

Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. CFPS can be applied for high-throughput screening of target proteins [7,8], functional tool for proteomics [7,9], and the development of point-of-care medicines [10,11]. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS [13]. Common secondary energy compounds such as creatine phosphate, phosphoenolpyruvate (PEP), acetate phosphate, glucose-6-phosphate (G6P), 3-phosphoglycerate, and fructose-1,6-biphosphate, contain high-energy phosphate bonds for regenerating ATP. ATP regeneration accompanied with the consumption of phosphate-containing substrate leads to an increase in inorganic phosphate. High concentrations of inorganic (page number not for citation purposes)

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Conclusion