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 alpha-amylase was used as a model protein. In the chemostat experiments, the total alpha-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 alpha-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 alpha-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%.