Systems for the Cell-Free Synthesis of Proteins

Abstract We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli . Two reaction con“gurations, batch and continuous exchange, are discussed and analytical scale as well as pre-parative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide speci“c protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exempli“ed as new tools for genomics and proteomics studies.Key words: Cell-free expression, Membrane proteins, Expression optimization, Detergents, Liposomes, Cell extracts Cell-free (CF) expression systems have emerged as important tools for the production of proteins at high levels ( 1 ). Besides general applicability to the production and labeling of proteins, these tech-niques are particularly successful for producing dif“cult proteins, such as integral membrane proteins (MPs), or toxic proteins. In conventional cellular systems, failure to overproduce MPs is fre-quently caused by overloading cellular transportation and translo-cation mechanisms, as well as by the toxicity of the synthesized proteins. CF expression generally eliminates these principal prob-lems and in addition, facilitates the downstream harvesting and isolation of MPs ( 2, 3 ). Furthermore, the open nature of CF reac-tions provides a valuable and unique way of directly modifying