Methodology article

Cell-free protein synthesis energized by slowly-metabolized maltodextrin

Yiran Wang¹ and Y-H Percival Zhang^1,2

¹Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA

²Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA

author email corresponding author email

BMC Biotechnology 2009, 9:58doi:10.1186/1472-6750-9-58

Published:	28 June 2009

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.