Expression-independent consumption of substrates in cell-free expression system from Escherichia coli

In a cell-free expression system derived from Escherichia coli, expression is abruptly ceased after 30 min of incubation while at this time not all th e substrates have been utilized in expression. Expression-independent consu mption of phosphoenolpyruvate and cysteine was found in this system, which was responsible for the above sudden cessation of expression. The above con sumption was at least partially due to the dephosphorylation of nucleoside triphosphates and the conversion of cysteine into gamma -glutamylcysteine, respectively. Based on these, we developed a new system employing new S30 e xtract of lower phosphatase activity, higher cysteine concentration, and an inhibitor of glutathione synthesis pathway. This system showed 70% enhance in productivity (179-302 mug chloramphenicolacetyltransferase protein per mi reaction mixture per hour) over the model system. (C) 2000 Elsevier Scie nce B.V. All rights reserved.