Prolonged cell‐free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate

Abstract

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

“…The utilization of phosphorylated energy donors for ATP regeneration results in excessive accumulation of inorganic phosphate. There have been speculations that phosphate might inhibit the translation reaction by complexing free Mg 2+ ions [12,14,31]; however, no causal mechanism has been proposed. Comparison of ribosome stability in two systems that differed in their ability to maintain a Mg 2+ homeostasis allowed us to link the observed 16S rRNA degradation directly to the availability of free Mg 2+ ions.…”

Development of an artificial cell, from self-organization to computation and self-reproduction

“…An artificial cell can in principle rely on the transcription/translation machinery provided by the extract for at least one generation. Important ongoing efforts are also made to improve the energy regeneration in cell-free reactions (31,32).…”

Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source

“…Cell-free protein synthesis reactions were conducted in 1.5 mL microtubes in a water bath set to 37 • C. The plasmid pK7CAT [12] encoding bacterial chloramphenicol acetyltransferase (CAT) under the control of the T7 promoter was used as the template for the cell-free protein synthesis reactions. The standard reaction mixture consisted of the following components: 57 mM Hepes-KOH (pH 8.2), 1.2 mM ATP, 0.85 mM each of CTP, GTP and UTP, 2 mM DTT, 0.17 mg/mL E. coli total tRNA mixture (from strain MRE600), 90 mM potassium glutamate, 80 mM ammonium acetate, 8 mM magnesium acetate, 20 mM potassium phosphate (pH 7.2), 34 g/mL L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid (folinic acid), 3.2 mM each of 20 amino acids, 2% (w/v) PEG (8000), 27 mM glucose, 10 M L-[U-14 C] leucine (11.3 GBq/mmol), 6.7 g/mL DNA, and 27% (v/v) S12 extract.…”

“…These advances have primarily been possible due to implementation of more efficient strategies for ATP regeneration during protein synthesis reactions. While the use of conventional energy sources (such as creatine phosphate, phosphoenolpyruvate and acetyl phosphate) has been limited by the high cost of reagents as well as the accumulation of inorganic phosphates [9], glycolytic intermediates can serve as an inexpensive and efficient energy source for cell-free protein synthesis in optimized cell extracts. More recently, Swartz and coworkers demonstrated that central carbon catabolism and oxidative phosphorylation reactions can be activated in cell extract for the supply of ATP during cell-free protein synthesis [5,7].…”