Chapter 2. Development of key technologies for high-throughput cell-free protein production with the extract from...

The cell-free translation system from wheat embryos had been considered to be inefficient as compared with the E. coli cell-based and cell-free protein production methods. However, it was revealed that the extract from extensively washed wheat embryo particles can provide a very productive cell-free protein synthesis system. Since then, the method has been improved, so that it fits the postgenomic researches. New mRNA configurations enabled us to synthesize many different proteins in parallel and to prepare large amounts of proteins, which fits the need for screening of suitable proteins for structural and functional analyses before large-scale production. The new reaction formats promoted the developments of new machines that perform highly parallel and highly productive protein synthesis reactions automatically. It was revealed that, by parallel synthesis of many proteins, much more multidomain proteins are produced in soluble forms in the wheat system than in the prokaryotic systems. The wheat system provides a rapid and cost-effective method for stable isotope labeling of proteins for NMR analyses. Selenomethionine substitution of proteins for X-ray crystallography through the cell-free synthesis was also achieved. Synthesis of some families of proteins that were difficult to be produced by conventional methods has been tested. At least, cytotoxic restriction enzymes were readily produced in a large amount. Some multisubunit proteins and cofactor-binding proteins could be synthesized by the method and were characterized successfully. Membrane proteins have also been tested, and a transporter was synthesized in an active form. Although some issues remains to be solved, we expect that the wheat cell-free protein synthesis system can contribute to the structural and functional genomics and to the future understanding of life.