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Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4214-6991
2005 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 242, no 1, 73-79 p.Article in journal (Refereed) Published
Abstract [en]

The TEM-1 beta-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 P-lactamase complementation in vitro with the fluorescent P-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 P-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.

Place, publisher, year, edition, pages
2005. Vol. 242, no 1, 73-79 p.
Keyword [en]
Escherichia coli, beta-lactamase, protein fragment complementation, affibody, CCF2/AM, fluorescence, protein-protein interactions, bacterial receptor domain, mammalian-cells, in-vivo, complementation, selection, display, libraries, reporter, cutinase
Identifiers
URN: urn:nbn:se:kth:diva-14457DOI: 10.1016/j.femsle.2004.10.047ISI: 000226264100010Scopus ID: 2-s2.0-11144312470OAI: oai:DiVA.org:kth-14457DiVA: diva2:332498
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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Nygren, Per-Åke

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