| Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease. | |
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MedLine Citation:
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PMID: 17586456 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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 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. |
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Authors:
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Richard H Kimura; Erin R Steenblock; Julio A Camarero |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S. Date: 2007-05-18 |
Journal Detail:
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Title: Analytical biochemistry Volume: 369 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 2007 Oct |
Date Detail:
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Created Date: 2007-09-03 Completed Date: 2007-12-13 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: United States |
Other Details:
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Languages: eng Pagination: 60-70 Citation Subset: IM |
Affiliation:
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Biosciences and Biotechnology Division, Livermore National Laboratory, University of California, Livermore, CA 94550, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Antigens, Bacterial / chemistry*, genetics, metabolism Bacillus anthracis / chemistry, enzymology* Bacterial Proteins / chemistry, genetics, metabolism* Bacterial Toxins / chemistry*, genetics, metabolism Biosensing Techniques Escherichia coli / genetics Fluorescence Resonance Energy Transfer / methods* Genes, Reporter Green Fluorescent Proteins / chemistry, genetics Luminescent Proteins / chemistry, genetics Models, Biological Molecular Sequence Data Oligopeptides / chemistry, metabolism Peptide Hydrolases / chemistry, genetics, metabolism* Protein Engineering Recombinant Fusion Proteins / chemistry, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Antigens, Bacterial; 0/Bacterial Proteins; 0/Bacterial Toxins; 0/Cyan Fluorescent Protein; 0/Luminescent Proteins; 0/Oligopeptides; 0/Recombinant Fusion Proteins; 0/anthrax toxin; 0/yellow fluorescent protein, Bacteria; 147336-22-9/Green Fluorescent Proteins; EC 3.4.-/Peptide Hydrolases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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