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

在活细胞中表达外源基因通常面临许多限制。产生的蛋白质可能在细胞内不稳定，甚至对宿主细胞有毒性。在活细胞中合成的蛋白质中通常很难引入非天然氨基酸。如果在无细胞系统中翻译是可能的，这样的限制是可以避免的。然而，目前可用的不同版本的无细胞系统都存在蛋白质产品产量低的问题。一些在无细胞系统中合成的蛋白质很难折叠成天然构象，因为分子伴侣的存在稳定了蛋白质的未折叠状态。在本项目中，我们的目标是开发一种无细胞翻译系统，该系统具有(1)提高产量(例如，高达10-100倍)和(2)提高生产功能蛋白的能力。为了提高蛋白质合成的产量，我们通过优化反应混合物的组成等方法，获得了每1毫升反应混合物中高达250杯蛋白质的产量。为了提高生产功能蛋白质的能力，我们已经建立了控制细胞提取物中分子伴侣数量的方法，用于体外蛋白质合成。例如，我们可以从酵母细胞提取物中去除85%的SSA蛋白(胞质HSP70)或100%的Ydjlp(胞质DNAJ同源物)进行无细胞翻译。虽然这些分子伴侣的缺失降低了蛋白质合成的产率，但它仍然使我们能够表征产生的蛋白质的构象/功能。

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.