GFP-visualized immobilized enzymes: degradation of paraoxon via organophosphorus hydrolase in a packed column.
A versatile gene-fusion technique for immobilizing and visualizing biologically active enzymes which includes from the N to C-termini, an affinity histidine tag, the green fluorescent protein (GFP), a proteolytic enzyme (enterokinase, EK) cleavage site and the enzyme of interest, were developed. Specifically, the organophosphorus hydrolase was bound to the affinity (His(6))-reporter(GFP)-EK fusion elements. Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. In the case of immobilized OPH, paraoxon was rapidly degraded when pumped through a packed column. In reaction mixtures containing CHES buffer at pH 6.9, a continual decay in OPH activity was observed and importantly, this was monitored by GFP fluorescence. This decay in activity was fully restored, along with fluorescence, upon washing with PBS buffer. Many subsequent experiments were performed at varied pH and in different background buffer solutions. In all cases when there was OPH activity there was also marked fluorescence from the GFP fusion partner. Likewise, when OPH activity was lost, so was GFP fluorescence and, importantly, both were regenerated when washed in the presence of the kosmotropic salt, phosphate. Recently, Waldo et al. (1999) showed that GFP fluorescence from whole cells indicated the extent of proper folding of normally aggregated proteins designed via directed evolution. The present work demonstrates an application wherein GFP fluorescence indicates stability and activity of its fusion partner.
Wu
Chi-Fang
CF
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, University of Maryland, College Park, Maryland 20742, USA.
Cha
Hyung Joon
HJ
Valdes
James J
JJ
Bentley
William E
WE
D043303:Aryldialkylphosphatase; D001483:Base Sequence; D017931:DNA Primers; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D007306:Insecticides / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D010261:Paraoxon / Q000378:metabolism*
D017931:DNA Primers; D004800:Enzymes, Immobilized; D007306:Insecticides; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases; D043303:Aryldialkylphosphatase; D010261:Paraoxon