Construction of an efficient Escherichia coli cell-free system for in vitro expression of several kinds of proteins

Cell-free protein synthesis systems have offered several advantages over traditional cell-based expression methods. In this study, the effects of extract preparation and an energy-regenerating system on protein synthesis were investigated in an Escherichia coli cell-free system. The results indicated that the expression level of enhanced green fluorescent protein (eGFP) with the S12 extract was higher than that with the S30 extract. Among four adenosine triphosphate-regenerating sources, the cAMP/CP/CK system (including cAMP, creatine phosphate, and creatine kinase) proved to be the most efficient one to support high-level expression of eGFP. Further studies showed that this established cell-free system could be successfully used to produce one model protein (eGFP), two human proteins (AK2 and coenzyme synthase) and two membrane proteins (subunit b of F1F0 adenosine triphosphate synthase and aquaporin Z). This outcome will be helpful to develop the highly efficient cell-free technology for the production of various proteins with different bio-origins.