Open AccessFluorescent detection of β‐lactamase activity in living Escherichia coli cells via esterase supplementation
Author(s) -
Nord Olof,
Gustrin Anna,
Nygren PerÅke
Publication year - 2005
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2004.10.047
Subject(s) - escherichia coli , periplasmic space , cutinase , esterase , protein fragment complementation assay , complementation , biochemistry , chemistry , biology , microbiology and biotechnology , enzyme , phenotype , gene
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.