Development of a cell-free translation system capable of producing functional proteins in high yield.

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Expression of alien genes in living cells often faces a number of limitations. The produced proteins may be unstable in cells, or even toxic to host cells. It is generally difficult to introduce unnatural amino acids into proteins synthesized in living cells. Such limitations could be avoided if translation were possible in cell-free systems. However different version of cell-free systems available today suffer from low yield of the protein product. Some proteins synthesized in a cell-free system hardly fold into native conformations because of the presence of molecular chaperones that stabilize unfolded states of the proteins. In the present project, we aimed at developing a cell-free translation system with (1) improved yield (e.g.as much as 10-100-fold) and (2) improved abilities to produce functional proteins.For improving the yield of protein synthesis, we attained yield of as much as 250 mug of proteins per 1 mL of a reaction mixture by optimizing the compositions of reaction mixtures etc.in the cell-free translation system with E.coli S30 extracts. For improving abilities to produce functional proteins, we have established methods to manipulate the amounts of molecular chaperones in the cell extracts for in vitro protein synthesis. For example, we could deplete 85% of Ssa proteins (cytosolic hsp 70) or 100% of Ydjlp (cytosolic DnaJ homolog) from yeast cell extracts for cell-free translation. Although depletion of these molecular chaperones decreased the yield of protein synthesis, but it still allowed us to characterize the conformation/functions of produced proteins.