E. coli S30 lysate proteome - The E

Protein production using processed cell lysates, defined precursor cocktails and target specific additives has become a core technology in the field of synthetic biology. Due to their open nature, these artificial systems are excellent to produce difficult proteins such as toxins or membrane proteins. However, the composition of cell lysates as the core component of cell-free systems is still widely a black box. Lysates of Escherichia coli are most productive for cell-free expression, yielding several milligrams of protein per milliliter of reaction. Although proteomics studies of several E. coli strains are available, the preparation of cell-free lysates implies specific fractionation resulting into yet unknown final lysate compositions. Therefore, the identification of prevalent and reliable lysate compounds will be an indispensable prerequisite for directed lysate engineering as an emerging strategy for new applications in synthetic biology. In this study, the S30 lysate proteome from E. coli strain A19 has been analyzed for the first time by using a GeLC-MS/MS approach. A total of 821 proteins were identified in processed S30 lysates. We provide detailed lists of detected proteins relevant for transcription/translation, folding, stability and enzymes involved in metabolic processes. In addition, we also performed a quantitative proteome analysis of modified S30 lysates, generated by induction of the SOS response during cultivation. These altered lysates contain 3-10-fold more folding factors and exhibit improved solubility and folding of difficult proteins. Therefore, the manipulation of the S30 lysate proteome by modified cultivation conditions is an effective approach for the generation of customized cell-free systems.