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Fluorescent detection of β-lactamase activity in living Escherichia coli cells via esterase supplementation
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Fluorescent detection of β-lactamase activity in living Escherichia coli cells via esterase supplementation
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Fluorescent detection of β-lactamase activity in living Escherichia coli cells via esterase supplementation
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Journal Article
Fluorescent detection of β-lactamase activity in living Escherichia coli cells via esterase supplementation
2005
Overview
The TEM-1 β-lactamase protein fragment complementation assay was investigated for its applicability in affinity protein-based interaction studies in
Escherichia coli, using an affibody-based model system. Results from co-transformation experiments showed that an ampicillin resistant phenotype was specifically associated with cognate affibody–target pairings. Attempts to monitor β-lactamase complementation in vitro with the fluorescent β-lactamase substrates CCF2/AM and CCF2 showed that
E. coli lacks an esterase activity necessary for activation of the esterified and membrane-permeable CCF2/AM form of the substrate. Interestingly, supplementation of the assay reaction with a purified fungal lipase (cutinase) resulted in efficient activation of CCF2/AM in vitro. Further, periplasmic expression of cutinase allowed for fluorescent discrimination between β-lactamase positive and negative living
E. coli cells using the CCF2/AM substrate, which should open the way for novel applications for this prokaryotic host in protein interaction studies.
Publisher
Elsevier B.V,Blackwell Publishing Ltd,Blackwell,Oxford University Press
Subject
/ Affibody
/ Bacteria
/ Biological and medical sciences
/ Carboxylic Ester Hydrolases - metabolism
/ CCF2/AM
/ Cutinase
/ display
/ E coli
/ Escherichia coli - enzymology
/ Escherichia coli Proteins - metabolism
/ Esterase
/ Fundamental and applied biological sciences. Psychology
/ Genetics
/ in-vivo
/ Kinases
/ Lipase
/ Protein fragment complementation
/ protein-protein interactions
/ Proteins
/ reporter
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