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 the substrates have been utilized in expression. Expression-independent consumption of phosphoenolpyruvate and cysteine was found in this system, which was responsible for the above sudden cessation of expression. The above consumption 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 extract of lower phosphatase activity, higher cysteine concentration, and an inhibitor of glutathione synthesis pathway. This system showed 70% enhance in productivity (179-302 microg chloramphenicolacetyltransferase protein per ml reaction mixture per hour) over the model system.