Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Cell-free coupled transcription-translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 +/- 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the...