Reconstitution of the Cpx signaling system from cell-free synthesized proteins

Cell-free expression has received growing attention as an effective system to produce integral membrane proteins for biochemical studies. We have applied this technology for the production of the histidine kinase CpxA, an integral membrane sensor that regulates an envelope stress response in Escherichia coli. All phosphotransfer activities of detergent-solubilized CpxA synthesized in vitro have been characterized and compared with those of CpxA solubilized from bacterial membranes. The results demonstrate the simplicity and efficiency of this technology for purifying large quantities of functional membrane proteins.