Preparation of a high expression yield bacterial cell extract via lysozyme-assisted sonication

One of the central goals for the development of cell-free (CF) gene expression systems is to ensure reproducible expression of proteins at high quality and yield. Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols have been developed and optimized reaction conditions have been established. One of the crucial steps during the preparation of cell extract-based expression systems, however, is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here, we demonstrate for an E. coli based system that a lysis procedure combining incubation of the cells with lysozyme with a gentle sonication step results in highly active cell extracts. As examples for the capabilities of our extract, we demonstrate the production of several fluorescent proteins as well as the production and assembly of T7 bacteriophages. Compared to other cell-free expression systems, our method achieved the highest phage titers in our plaque assays. Stateof-the-art quantitative proteomics revealed that enzymes of the energy regeneration pathway were enriched in our extract compared to a widely used commercial cell extract. On the other hand, ribosomal proteins were found to be more abundant in the commercial product.