Allen Michael S, Wilgus John R, Chewning Christopher S, Sayler Gary S, Simpson Michael L
Molecular-Scale Engineering and Nanoscale Technologies (MENT) Research Group, Oak Ridge National Laboratory, P.O. Box 2008, Building 3500, MS 6006, Oak Ridge, TN, 37931-6006, USA.
Syst Synth Biol. 2007 Mar;1(1):3-9. doi: 10.1007/s11693-006-9001-5.
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.
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，并已成为合成基因回路应用中的一个主要组成部分。许多这类系统的一个主要局限性是报告蛋白的半衰期相对较长，这使得无法实时监测转录的起始和终止。此外，当用作合成基因回路的组件时，与报告蛋白降解相关的长时间常数可能会显著降低回路性能。在本研究中，通过加入一个被内源性尾特异性蛋白酶识别的11个氨基酸的羧基末端标签，在大肠杆菌中构建了来自发光杆菌的LuxA和LuxB的短半衰期变体。结果表明，当标签添加到luxA或同时添加到luxA和luxB时，C末端标签的添加会影响全酶的功能半衰期，但仅对luxB进行修饰没有显著影响。此外，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了半衰期中等长度的变体，其方式与报道的绿色荧光蛋白类似。本报告是将C末端标签方法用于调节蛋白质半衰期首次应用于一种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在监测基因表达动态变化方面的应用。
