pHluorin-based in vivo assay for hydrolase screening.
pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.
Schuster
Sascha
S
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Enzelberger
Markus
M
Trauthwein
Harald
H
Schmid
Rolf D
RD
Urlacher
Vlada B
VB
D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D004950:Esterases / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D049408:Luminescent Agents / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence
D049408:Luminescent Agents; C485187:PHluorin; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases