使用重构的体外蛋白质合成系统合成毫克量的蛋白质。
Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.
作者信息
Kazuta Yasuaki, Matsuura Tomoaki, Ichihashi Norikazu, Yomo Tetsuya
机构信息
Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
出版信息
J Biosci Bioeng. 2014 Nov;118(5):554-7. doi: 10.1016/j.jbiosc.2014.04.019. Epub 2014 May 28.
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 0.38 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 β-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.
在本研究中，对仅由高度纯化的组分组成的体外蛋白质合成系统（PURE系统）合成蛋白质的量进行了优化。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0.38mg/mL绿色荧光蛋白（GFP）以及在透析模式下合成3.8mg/mL GFP时的组分浓度。在透析模式下，二氢叶酸还原酶和β-半乳糖苷酶的合成蛋白浓度分别为4.3mg/mL和4.4mg/mL。使用优化后的系统，合成的蛋白质占总蛋白的30%（w/w），这与大肠杆菌细胞中过表达蛋白的水平相当。这种优化的重组体外蛋白质合成系统可能潜在地用于各种应用，包括蛋白质的体外定向进化、人工细胞组装和蛋白质结构研究。
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