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Cell-Free Production of Aggregation-Prone Proteins in Soluble and Active Forms

机译：无细胞生产可溶性和活性形式的聚集蛋白

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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 endog-enously 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.
机译：我们已经开发了一种高效的无细胞蛋白质合成系统，用于生产可溶性和活性真核蛋白质，这些蛋白质主要以细菌的包涵体形式产生.S30提取物（以30,000g离心时表明细胞匀浆的上清液）表示无细胞蛋白质从大肠杆菌制备合成蛋白，将其修饰以过量表达一组伴侣蛋白（GroEL / ES或DnaK / J-GrpE）和二硫键异构酶（在细胞质中表达无前导序列的成熟DsbC）溶解度和生物活性浓度（生物新的无细胞蛋白质合成系统合成的蛋白质的每单位体积无细胞蛋白质合成反应混合物的活性显示出显着的改善.DnaK / J-GrpE的存在使溶解度的提高最为显着。折叠试验前与DnaK / J-GrpE的共翻译相互作用对维持折叠能力下易于凝集的蛋白质很重要为了最大化表达蛋白的生物活性浓度，还需要另外存在GroEL / ES和DsbC。当人类促红细胞生成素在已开发的无细胞蛋白合成系统中表达时，包括内源性过表达的伴侣蛋白和/或DsbC ，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改组以及共翻译折叠对于从无细胞表达系统获得功能活性蛋白至关重要。使用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合成中有效生产可溶性和活性蛋白的报道。这种新型的无细胞蛋白合成系统能够引入大量的伴侣蛋白和二硫键异构酶。相比于单独补充它们的常规方法。高效的无细胞蛋白质合成系统支持以可溶性和活性形式有效表达真核蛋白质，而无需折叠效应子的任何外源添加或共表达。

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