Continuous Production of Cell‐Free Recombinant Proteins Using Escherichia coli

Continuous production of extracellular recombinant protein using a previously reported periplasmic protein‐release system was successfully demonstrated. The performance of the protein‐releasing system was studied using two types of reactor configuration: a conventional chemostat and a chemostat with a cell‐recycle system. The constitutively expressed α ‐amylase was used as a model protein. In the chemostat experiments, the total α ‐amylase activity increases with decreasing dilution rate—from 135.5 units/mL at a dilution rate of 0.05 h −1 to 2.06 units/mL at a dilution rate of 0.491 h −1 . However, the percentage release of α ‐amylase remained relatively constant, at approximately 29%, in the range of dilution rates investigated. The reactor productivity of the target protein reached a maximum of 8.14 units/mL/h at a dilution rate of 0.1 h −1 . Application of the releasing system for the continuous production of cell‐free recombinant proteins in a cell‐recycle fermentor was also studied. The reactor performance is significantly better than that of either the chemostat or the batch mode. A total productivity of α ‐amylase at 13.2 units/mL/h with 42% recovery of proteins in the cell‐free form was obtained at a dilution rate of 0.397 h −1 and a bleeding rate of 9.8%.