title: Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease.
abstract: 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.
authors.affiliation: Biosciences and Biotechnology Division, Livermore National Laboratory, University of California, Livermore, CA 94550, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Erin R
authors.initials: ER
authors.lastname: Steenblock
authors.forename: Julio A
authors.initials: JA
authors.lastname: Camarero
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001408:Bacillus anthracis / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases