Development of a Cell-Based Fluorescence Resonance Energy Transfer Reporter for Bacillus anthracis Lethal Factor Protease

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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 ... continued below

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Kimura, R H; Steenblock, E R & Camarero, J A March 22, 2007.

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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 5 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.

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PDF-file: 31 pages; size: 1.8 Mbytes

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  • Journal Name: Analytical Biochemistry, vol. 369, no. 1, October 1, 2007, pp. 60-70; Journal Volume: 369; Journal Issue: 1

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  • Report No.: UCRL-JRNL-230670
  • Grant Number: W-7405-ENG-48
  • Office of Scientific & Technical Information Report Number: 940510
  • Archival Resource Key: ark:/67531/metadc896853

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Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

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  • March 22, 2007

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  • Sept. 27, 2016, 1:39 a.m.

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  • Nov. 23, 2016, 3:25 p.m.

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Kimura, R H; Steenblock, E R & Camarero, J A. Development of a Cell-Based Fluorescence Resonance Energy Transfer Reporter for Bacillus anthracis Lethal Factor Protease, article, March 22, 2007; Livermore, California. (digital.library.unt.edu/ark:/67531/metadc896853/: accessed December 16, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.