Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation.
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.
Nord
Olof
O
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Gustrin
Anna
A
Nygren
Per-Ake
PA
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D004950:Esterases / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D007769:Lactams / Q000378:metabolism; D011485:Protein Binding; D001618:beta-Lactamases / Q000032:analysis*
C109764:CCF 2; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D007769:Lactams; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1