Protein productivity of cell-free translation was improved by removing phosphatase from wheat germ extract with immunoprecipitation

Abstract

The biochemical energy charge, ([ATP] + [ADP]/2)/([ATP] + [ADP] + [AMP]), is necessary to be kept at a high level close to 1.0 for protein synthesis in a cell-free translation system. Aiming at improvement of protein productivity of wheat germ extract (WGE) cell-free translation system, the authors developed a method of maintaining the energy charge at the high level by removing acid phosphatase activity of hydrolyzing ATP from the system. About 3 unit/ml activity of acid phosphatase (ACP) was detected in WGE. The detected activity of ACP accounted for 90% of ATP consumption in WGE cell-free translation system. Immunoprecipitation of ACP with ACP-specific antiserum reduced ACP activity in WGE by 60%. As the result, the energy charge in the translation system was kept above 0.97 for 150 min when the antiserum-treated WGE was used, while it was kept so for only 40 min when the untreated WGE was used. The protein productivity of the translation system increased 5-fold by the immunoprecipitation of ACP. Affinity separation of ACP with cellulose phosphate, or inhibition of ACP activity with inhibitors such as molybdate and vanadate reduced ACP activity in the translation system. However, these non-biological methods resulted in decrease rather than increase in protein productivity.