Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease
Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease
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DOI：
10.1016/j.ab.2007.05.014
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发表时间：
2007-10-01
影响因子：
2.9
通讯作者：
Camarero, Julio A.
中科院分区：
文献类型：
--
作者：
Kimura, Richard H.;Steenblock, Erin R.;Camarero, Julio A.
我们报告的炭疽致死因子（LF）蛋白酶活性的细胞为基础的荧光报告分子的构建使用荧光共振能量转移（FRET）的原则。这是通过工程化大肠杆菌细胞系以表达遗传编码的FRET报告基因和LF蛋白酶来实现的。两种蛋白质在两种不同的表达质粒中编码，在不同的严格控制的诱导型启动子的控制下。基于FRET的报告基因被设计为含有LF识别序列，其侧翼是由CyPet和YPet荧光蛋白形成的FRET对。使用含有几个Gly-Gly-Ser重复序列的柔性肽接头优化两种荧光蛋白之间的接头长度。我们的研究结果表明，这种基于FRET的LF报告基因在大肠杆菌中很容易表达。大肠杆菌细胞显示高水平的FRET在体内的LF的情况下。然而，在同一细胞中诱导LF表达后，FRET信号降低了5倍。这些结果表明，这种基于细胞的LF FRET报告基因可用于筛选体内抗LF的遗传编码文库。(c)2007年爱思唯尔公司All rights reserved.
We report the construction of a cell-based fluorescent reporter for anthrax lethal factor (LF) protease activity using the principle of fluorescence resonance energy transfer (FRET). This was accomplished by engineering an Escherichia coli cell line to express a genetically encoded FRET reporter and LF protease. Both proteins were encoded in two different expression plasmids under the control of different tightly controlled inducible promoters. The FRET-based reporter was designed to contain a LF recognition sequence flanked by the FRET pair formed by CyPet and YPet fluorescent proteins. The length of the linker between both fluorescent proteins was optimized using a flexible peptide linker containing several Gly-Gly-Ser repeats. Our results indicate that this FRET-based LF reporter was readily expressed in E. coli cells showing high levels of FRET in vivo in the absence of LF. The FRET signal, however, decreased five times after inducing LF expression in the same cell. These results suggest that this cell-based LF FRET reporter may be used to screen genetically encoded libraries in vivo against LF. (c) 2007 Elsevier Inc. All rights reserved.