Document Detail


Novel fluorescence-assisted whole-cell assay for engineering and characterization of proteases and their substrates.
MedLine Citation:
PMID:  20851955     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.
Authors:
George Kostallas; Patrik Samuelson
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-09-17
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  76     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-11-08     Completed Date:  2011-02-22     Revised Date:  2011-07-28    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  7500-8     Citation Subset:  IM    
Affiliation:
Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
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MeSH Terms
Descriptor/Qualifier:
Escherichia coli / genetics,  metabolism
Fluorescence*
Peptide Hydrolases / genetics*,  metabolism*
Potyvirus / enzymology
Protein Engineering / methods*
Recombinant Proteins / genetics,  metabolism
Staining and Labeling / methods*
Viral Proteins / genetics,  metabolism
Chemical
Reg. No./Substance:
0/Recombinant Proteins; 0/Viral Proteins; EC 3.4.-/Peptide Hydrolases
Comments/Corrections

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