Crosstalk between Diverse Synthetic Protein Degradation Tags in Escherichia coli,ACS Synthetic Biology

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Crosstalk between Diverse Synthetic Protein Degradation Tags in Escherichia coli
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-12-13 00:00:00 , DOI: 10.1021/acssynbio.7b00122
Nicholas C. Butzin ₁ , William H. Mather ₂

Affiliation

Recently, a synthetic circuit in E. coli demonstrated that two proteins engineered with LAA tags targeted to the native protease ClpXP are susceptible to crosstalk due to competition for degradation between proteins. To understand proteolytic crosstalk beyond the single protease regime, we investigated in E. coli a set of synthetic circuits designed to probe the dynamics of existing and novel degradation tags fused to fluorescent proteins. These circuits were tested using both microplate reader and single-cell assays. We first quantified the degradation rates of each tag in isolation. We then tested if there was crosstalk between two distinguishable fluorescent proteins engineered with identical or different degradation tags. We demonstrated that proteolytic crosstalk was indeed not limited to the LAA degradation tag, but was also apparent between other diverse tags, supporting the complexity of the E. coli protein degradation system.

中文翻译：

大肠杆菌中各种合成蛋白降解标签之间的串扰

最近，在大肠杆菌中的合成电路表明，针对天然蛋白酶ClpXP靶向LAA标签的两种蛋白质，由于竞争蛋白质之间的降解而易受串扰的影响。为了了解超越单一蛋白酶机制的蛋白水解串扰，我们在大肠杆菌中进行了研究一组合成电路，旨在探测与荧光蛋白融合的现有和新型降解标签的动力学。使用酶标仪和单细胞分析法对这些电路进行了测试。我们首先单独量化每个标签的降解率。然后，我们测试了用相同或不同降解标签改造的两个可区分的荧光蛋白之间是否存在串扰。我们证明了蛋白水解串扰确实不仅仅限于LAA降解标签，而且在其他各种标签之间也很明显，这支持了大肠杆菌蛋白质降解系统的复杂性。

更新日期：2017-12-13

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