Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system
Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system
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DOI：
10.1016/j.jbiosc.2014.04.019
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发表时间：
2014-11-01
影响因子：
2.8
通讯作者：
Yomo, Tetsuya
中科院分区：
文献类型：
--
作者：
Kazuta, Yasuaki;Matsuura, Tomoaki;Yomo, Tetsuya
在本研究中，优化了使用仅由高度纯化的组分组成的体外蛋白质合成系统（PURE系统）合成的蛋白质的量。通过改变每个系统组分的浓度，我们确定了在分批模式下合成038 mg/mL绿色荧光蛋白（GFP）和在透析模式下合成3.8 mg/mL GFP的组分浓度。在透析模式下，二氢叶酸还原酶和β-半乳糖苷酶的蛋白质浓度分别为4.3和4.4 mg/mL。使用优化的系统，合成的蛋白质占总蛋白质的30%（w/w），这与大肠杆菌细胞中过表达蛋白质的水平相当。这种优化的重组体外蛋白质合成系统可能是有用的各种应用，包括在体外定向进化的蛋白质，人工细胞组装和蛋白质结构的研究。(C)2014年，生物技术学会，日本。All rights reserved.
In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 038 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and beta-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.