Principles and applications of cell-free protein synthesis systems scale-up

Cell-free protein synthesis systems have many advantages over conventional rDNA protein expression technologies. Protein synthesis time is short, reaction conditions (such as pH and redox potential) can be easily controlled, and non-native substrates and catalysts can be precisely added to facilitate protein expression. These features make the cell-free systems well-suited for applications including personalized medicine, high throughput protein synthesis, unnatural amino-acid incorporation, and the expression of toxic proteins, as well as the study of complex cellular processes. Even though substantial progress has recently been made in improving the biochemistry of the cell-free systems, the adoption of the in vitro protein synthesis technology as an alternative to in vivo expression has been impeded by the inability to efficiently and rapidly increase the reaction volume. In this work we describe the principles and applications of new methods for the efficient scale-up of cell-free protein synthesis systems in batch mode suitable for any reaction volume without any reduction in protein synthesis yields. With the first method, the cell-free protein synthesis reactions are conducted in a thin film on a planar hydrophobic surface. The second method improves on the thin film approach by using a stirred tank reactor. We apply these methods to the development of vaccines against B-cell lymphoma and to the large scale production of human insulin-like growth factor 1 (IGF-1). The thin film approach is used to scale-up vaccine candidate production for in vivo studies. We show that the cell-free system can, rapidly and efficiently, produce patient-specific vaccines that offer protection against tumor challenge in a mouse animal model. The stirred tank reactor approach is used to produce 386 mg of soluble and properly folded IGF-1 in a 1 L cell free reaction using a conventional cell culture fermenter. Additionally, we demonstrate that the stirred tank reactor is a convenient platform for studying oxidative phosphorylation in cell-free systems. In summary, the work presented in this thesis furthers our understanding of in vitro protein expression technology and significantly enhances the utility of the cell-free system, both as a research tool and as an industrial platform for rapid and efficient protein production.