A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus 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 S. solfataricus 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 S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), 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.

中文翻译：

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基于Sulfolobus solfataricus细胞裂解物的体外转录/翻译系统的优化。

描述了一种允许在高温下体外有效合成蛋白质的系统。它基于使用以前在我们实验室中很好表征的Sulfolobus solfataricus的未分离细胞裂解液（S30）进行预转录的mRNA的翻译，现在可以进行偶联的转录和翻译。该表达系统中的必需元件是源自S.solfataricus 16S / 23S rRNA编码基因的强启动子，可以从该基因高效转录特定的mRNA。报告了两种不同蛋白质的合成，包括S. solfataricus DNA-烷基鸟嘌呤-DNA-烷基转移酶蛋白（SsOGT），已成功用适当的荧光底物标记并在细胞提取物中可见。实验方法的简单性和蛋白质的比活性为研究蛋白质的结构-功能关系提供了许多可能性。