Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP) regeneration systems: creatine phosphate (CP), phosphoenolpyruvate (PEP), and 3-phosphoglyceric acid (3-PGA). The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture.Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro.Cell-free systems are used to synthesize large amounts of recombinant proteins in a few hours. Cell-free expression, which has many advantages in comparison to cell-based expression, is employed in an increasing number of biotechnology and proteomic applications [1]. Many efforts are made to increase the protein productivity and the functionality of cell-free systems. The energy regeneration is frequently optimized [2-6], new E. coli strains are engineered to stabilize some amino acids or to express proteins with PCR products [7,8] and preparation of the crude extract is simplified [9]. Current extracts prepared from E. coli cells can