Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation

FEMS Microbiol Lett. 2005 Jan 1;242(1):73-9. doi: 10.1016/j.femsle.2004.10.047.


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 beta-lactamase complementation in vitro with the fluorescent beta-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 beta-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carboxylic Ester Hydrolases / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / metabolism
  • Esterases / metabolism*
  • Fluoresceins / metabolism
  • Fluorescence
  • Lactams / metabolism
  • Protein Binding
  • beta-Lactamases / analysis*


  • CCF 2
  • Escherichia coli Proteins
  • Fluoresceins
  • Lactams
  • Esterases
  • Carboxylic Ester Hydrolases
  • cutinase
  • beta-Lactamases
  • beta-lactamase TEM-1