Analysis of protein functions through a bacterial cell-free protein expression system

Cell-free protein synthesis is a suitable protein expression method for the high-throughput use because a PCR-amplified linear DNA fragment is utilized as a template for protein synthesis without any cloning procedures. We have developed a two-step PCR method for high-throughput and robust production of linear templates ready for cell-free protein synthesis. A high-throughput protein expression method has been established by combining the batch-mode cell-free protein synthesis with the two-step PCR, which is performed on multiwell plates, and is thus adapted for robotics. In this chapter, our two-step PCR method and the batch-mode cell-free protein synthesis are described.