Kimura Richard H, Steenblock Erin R, Camarero Julio A
Biosciences and Biotechnology Division, Livermore National Laboratory, University of California, Livermore, CA 94550, USA.
Anal Biochem. 2007 Oct 1;369(1):60-70. doi: 10.1016/j.ab.2007.05.014. Epub 2007 May 18.
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.
我们报告了利用荧光共振能量转移（FRET）原理构建用于炭疽致死因子（LF）蛋白酶活性的基于细胞的荧光报告系统。这是通过构建一种大肠杆菌细胞系来实现的，该细胞系可表达基因编码的FRET报告蛋白和LF蛋白酶。这两种蛋白分别由两个不同的表达质粒编码，并受不同的严格可控诱导型启动子控制。基于FRET的报告蛋白设计为包含一个LF识别序列，两侧是由CyPet和YPet荧光蛋白形成的FRET对。使用含有多个Gly-Gly-Ser重复序列的柔性肽接头优化了两种荧光蛋白之间的接头长度。我们的结果表明，这种基于FRET的LF报告蛋白在大肠杆菌细胞中易于表达，在无LF的情况下在体内显示出高水平的FRET。然而，在同一细胞中诱导LF表达后，FRET信号降低了五倍。这些结果表明，这种基于细胞的LF FRET报告系统可用于在体内针对LF筛选基因编码文库。
