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

Kimura, R H; Steenblock, E R; Camarero, J A

doi:10.1016/j.ab.2007.05.014

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

Journal Article · Thu Mar 22 00:00:00 EDT 2007 · Analytical Biochemistry

DOI:https://doi.org/10.1016/j.ab.2007.05.014· OSTI ID:940510

Kimura, R H; Steenblock, E R; Camarero, J A

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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Research Organization:: Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: W-7405-ENG-48

OSTI ID:: 940510

Report Number(s):: UCRL-JRNL-230670; ANBCA2; TRN: US200824%%73

Journal Information:: Analytical Biochemistry, Vol. 369, Issue 1; ISSN 0003-2697

Country of Publication:: United States

Language:: English

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

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