A destabilized bacterial luciferase for dynamic gene expression studies.
A destabilized bacterial luciferase for dynamic gene expression studies.
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DOI：
10.1007/s11693-006-9001-5
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发表时间：
2007-03-01
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影响因子：
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通讯作者：
Simpson, Michael L
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文献类型：
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作者：
Allen, Michael S;Wilgus, John R;Simpson, Michael L
基因调控元件与报告基因的融合长期以来一直被用作监测基因表达的工具，并已成为合成基因电路实现的主要组成部分。许多这些系统的一个主要限制是报告蛋白的半衰期相对较长，这阻碍了实时监测转录的起始和终止。此外，当作为合成基因电路的组成部分时，与报告蛋白衰变相关的长时间常数可能会显著降低电路的性能。在这项研究中，我们在大肠杆菌中构建了来自光habdus luminesens的LuxA和LuxB的短半衰期变体，通过包含一个11个氨基酸的羧基末端标签，该标签可以被内源性尾部特异性蛋白酶识别。结果表明，添加c端标签对luxA或同时添加luxA和luxB的全酶的功能半衰期有影响，而单独修饰luxB对全酶的功能半衰期没有显著影响。此外，研究还发现，与LuxA融合的羧基末端标签的末端三个氨基酸残基的改变产生的变体具有中等长度的半衰期，与报道的GFP相似。该报告是c端标记方法用于蛋白质半衰期调控的第一个实例，应用于酶或多亚基酶复合物的单体，并将扩展细菌荧光素酶报告基因用于监测基因表达动态变化的效用。
Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression.