Document Detail


Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease.
MedLine Citation:
PMID:  17586456     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Richard H Kimura; Erin R Steenblock; Julio A Camarero
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2007-05-18
Journal Detail:
Title:  Analytical biochemistry     Volume:  369     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2007 Oct 
Date Detail:
Created Date:  2007-09-03     Completed Date:  2007-12-13     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  60-70     Citation Subset:  IM    
Affiliation:
Biosciences and Biotechnology Division, Livermore National Laboratory, University of California, Livermore, CA 94550, USA.
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MeSH Terms
Descriptor/Qualifier:
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:
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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