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We compared the abilities of several ATP regeneration systems to support the production of soluble proteins in a cell-free synthesis system. While changes in the ATP level in the reaction mixture were variable depending on the ATP regeneration system employed, the yields and solubilities of synthesized proteins did not always correlate with the ATP level. Instead, the efficiency of cell-free protein synthesis was substantially affected by changes in the pH of the reaction mixture. These results suggest that measures to maintain the pH of the reaction mixture should be considered when choosing an energy source for cell-free protein synthesis.