A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins
A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins
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DOI：
10.1016/j.pep.2008.09.002
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发表时间：
2008-12-01
影响因子：
1.6
通讯作者：
Imataka, Hiroaki
中科院分区：
文献类型：
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作者：
Mikami, Satoshi;Kobayashi, Tominari;Imataka, Hiroaki
本研究的目的是建立一个高效的无细胞蛋白表达系统。我们用T7 RNA聚合酶和含有T7启动子/终止子单元的质粒建立了一个基于HeLa细胞的体外偶联转录/翻译系统。为了增强偶联系统中的蛋白质合成，我们将脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES）或丙型肝炎病毒（HCV）IRES置于质粒的T7启动子和编码区之间。值得注意的是，我们发现这些IRES依赖性系统能够产生包括GCN 2（160 kD）、Dicer（200 kD）和mTOR（260 kD）在内的大蛋白，达到通过考马斯亮蓝染色在SDS-PAGE上可检测的水平。我们将合成的蛋白质纯化至接近同质，并在适当的生化测定中验证其功能。总之，使用EMCV或HCV IRES的基于HeLa细胞的体外偶联转录/翻译系统是一种方便的工具，特别是用于生产大型重组蛋白。(c)2008年爱思唯尔公司All rights reserved.
The aim of this study was to develop an efficient cell-free protein expression system derived from mammalian cells. We established a HeLa Cell-based in vitro coupled transcription/translation system with T7 RNA polymerase and a plasmid that harbored a T7 promoter/terminator unit. To enhance protein synthesis in the coupled system, we placed the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or the hepatitis C virus (HCV) IRES between the T7 promoter and the coding region of the plasmid. Remarkably, we found that these IRES-dependent systems were able to produce large proteins including GCN2 (160 kD), Dicer (200 kD) and mTOR (260 kD) to levels detectable on SDS-PAGE by Comassie Brilliant Blue-staining. We purified the synthesized proteins to near homogeneity, and validated their functionalities in the appropriate biochemical assays. In conclusion, the HeLa cell-based in vitro coupled transcription/translation system using the EMCV or HCV IRES is a convenient tool, particularly for the production of large recombinant proteins. (c) 2008 Elsevier Inc. All rights reserved.