Lo Gullo Giada, Mattossovich Rosanna, Perugino Giuseppe, La Teana Anna, Londei Paola, Benelli Dario
Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Via Regina Elena 324, 00161 Rome, Italy.
Institute of Biosciences and BioResources, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples, Italy.
Archaea. 2019 Feb 11;2019:9848253. doi: 10.1155/2019/9848253. eCollection 2019.
A system is described which permits the efficient synthesis of proteins at high temperature. It is based on the use of an unfractionated cell lysate (S30) from previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the DNA-alkylguanine-DNA-alkyl-transferase protein (OGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.
本文描述了一种能够在高温下高效合成蛋白质的系统。该系统基于使用来自我们实验室先前已充分表征的未分级细胞裂解物（S30）来翻译预先转录的mRNA，并且现在已被改造用于进行耦合转录和翻译。这个表达系统的关键要素是一个源自16S/23S rRNA编码基因的强启动子，从该启动子可以高效转录特定的mRNA。报道了两种不同蛋白质的合成，包括DNA-烷基鸟嘌呤-DNA-烷基转移酶蛋白（OGT），该蛋白已被证明能用合适的荧光底物成功标记并在细胞提取物中可视化。实验过程的简单性和蛋白质的比活性为研究蛋白质的结构-功能关系提供了多种可能性。
