Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate.

IF 2.3 4区生物学 Q3 MICROBIOLOGY Archaea-An International Microbiological Journal Pub Date : 2019-02-11 eCollection Date: 2019-01-01 DOI:10.1155/2019/9848253

Giada Lo Gullo, Rosanna Mattossovich, Giuseppe Perugino, Anna La Teana, Paola Londei, Dario Benelli

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引用次数: 12

Abstract

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.

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基于硫酸根细胞裂解液的体外转录/翻译系统优化

本文描述了一种能在体外高温下高效合成蛋白质的系统。它是基于使用来自Sulfolobus solfataricus的未分离细胞裂解物(S30)，之前在我们的实验室中很好地表征了预转录mrna的翻译，现在适合于执行耦合转录和翻译。该表达系统的核心元件是来自于S. solfataricus 16S/23S rrna编码基因的强启动子，该启动子可高效转录特定mrna。报道了两种不同蛋白的合成，其中包括S. solfataricus dna -烷基鸟嘌呤- dna -烷基转移酶蛋白(SsOGT)，该蛋白被证明可以用适当的荧光底物成功标记，并在细胞提取物中可见。实验过程的简单性和蛋白质的特异性活性为研究蛋白质的结构-功能关系提供了许多可能性。

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来源期刊

Archaea-An International Microbiological Journal MICROBIOLOGY-

CiteScore

7.50

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： Archaea is a peer-reviewed, Open Access journal that publishes original research articles as well as review articles dealing with all aspects of archaea, including environmental adaptation, enzymology, genetics and genomics, metabolism, molecular biology, molecular ecology, phylogeny, and ultrastructure. Bioinformatics studies and biotechnological implications of archaea will be considered. Published since 2002, Archaea provides a unique venue for exchanging information about these extraordinary prokaryotes.

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