Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes
Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes
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DOI：
10.1016/j.jbiotec.2007.08.008
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发表时间：
2008-01-20
影响因子：
4.1
通讯作者：
Baba, Yoshinobu
中科院分区：
文献类型：
--
作者：
Hino, Mami;Kataoka, Masatoshi;Baba, Yoshinobu
甘油醛-3-磷酸脱氢酶（G3 PDH）的蛋白质合成的效率进行了研究，在几个体外耦合的转录/翻译蛋白质合成系统的基础上大肠杆菌裂解物，小麦胚芽，或网织红细胞裂解物，并在体外翻译系统的基础上小麦胚芽提取物。只有在基于E.以pET/G3 PDH为表达载体，在大肠杆菌中进行表达。用含有5 ′-珠蛋白前导序列和人工合成的poly（A）（30）尾的pT（N）T表达载体代替pET，在小麦胚中获得了显著的蛋白质合成增加。在前四个系统中未观察到通过Western印迹分析的T7 RNA聚合酶存在的显著差异，并且通过北方印迹分析的每个反应混合物中总RNA存在的差异似乎与蛋白质合成无关。虽然通过体外翻译系统使用用pET/G3 PDH体外转录的RNA编码G3 PDH合成少量蛋白质，但使用含有T7 RNA启动子和翻译增强子Omega序列的pEU/G3 PDH转录的RNA观察到极端增加。这些结果表明，翻译增强子序列的存在是真核无细胞蛋白质合成系统合成蛋白质的关键步骤之一。(c)2007 Elsevier B. V.保留所有权利。
The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT(N)T expression vector which contains a 5'-globin leader sequence and a synthetic poly(A)(30) tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Omega sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system. (c) 2007 Elsevier B.V. All rights reserved.