Kang Sang-Hyeon, Kim Dong-Myung, Kim Hyo-Jin, Jun Soo-Youn, Lee Ki-Young, Kim Hye-Jin
DreamBiogen Co., Ltd, 2F, Chungui Bldg., 238-4 Poi-dong, Gangnam-gu, Seoul 135-260, Korea.
Biotechnol Prog. 2005 Sep-Oct;21(5):1412-9. doi: 10.1021/bp050087y.
We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.
我们开发了一种高效的无细胞蛋白质合成系统，用于生产主要在细菌中以包涵体形式产生的可溶性和活性真核蛋白质。用于无细胞蛋白质合成的S30提取物（表示在30,000g离心时细胞匀浆的上清液）是从经修饰以过表达一组伴侣蛋白（GroEL/ES或DnaK/J-GrpE）和二硫键异构酶（在细胞质中表达的无前导序列的成熟DsbC）的大肠杆菌中制备的。新的无细胞蛋白质合成系统合成的蛋白质的溶解度和生物活性浓度（无细胞蛋白质合成反应混合物每单位体积的生物活性）有了显著提高。在存在DnaK/J-GrpE的情况下，溶解度增强最为显著。这表明在折叠试验之前与DnaK/J-GrpE的共翻译相互作用对于将易于聚集的蛋白质维持在具有折叠能力的可溶状态很重要。为了使表达蛋白质的生物活性浓度最大化，还需要额外存在GroEL/ES和DsbC。当在包括内源性过表达伴侣蛋白和/或DsbC的已开发无细胞蛋白质合成系统中表达人促红细胞生成素时，促红细胞生成素的生物活性浓度提高了700%。这意味着翻译后折叠和二硫键重排以及共翻译折叠对于从无细胞表达系统中获得功能活性蛋白质很重要。这是首次报道使用包括内源性过表达伴侣蛋白和/或二硫键异构酶的S30提取物在无细胞蛋白质合成中高效生产可溶性和活性蛋白质。与单独补充它们的传统方法相比，这种新的无细胞蛋白质合成系统能够引入大量的伴侣蛋白和二硫键异构酶。所开发的无细胞蛋白质合成系统支持以可溶性和活性形式高效表达真核蛋白质，而无需任何外源添加或折叠效应物的共表达。
