A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.
Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.
Wu
C F
CF
Center for Agricultural Biotechnology, University of Maryland, Biotechnology Institute, College Park 20742, USA.
Cha
H J
HJ
Rao
G
G
Valdes
J J
JJ
Bentley
W E
WE
D043303:Aryldialkylphosphatase; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases; D043303:Aryldialkylphosphatase