Cell-free protein synthesis energized by slowly-metabolized maltodextrin.
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.


CONCLUSIONS
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.
Wang
Yiran
Y
Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA. yiran@vt.edu
Zhang
Y-H Percival
YH
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000449:Aldehyde Reductase / Q000096:biosynthesis; D002474:Cell-Free System; D004950:Esterases / Q000096:biosynthesis; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism; D006019:Glycolysis; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D010733:Phosphoglucomutase / Q000378:metabolism; D010766:Phosphorylation; D011134:Polysaccharides / Q000378:metabolism*; D014176:Protein Biosynthesis
D005958:Glucosephosphates; D011134:Polysaccharides; D049452:Green Fluorescent Proteins; C008315:maltodextrin; D000255:Adenosine Triphosphate; C031590:glucose-1-phosphate; D000449:Aldehyde Reductase; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase; D004950:Esterases; D010733:Phosphoglucomutase