Orientation specific immobilization of organophosphorus hydrolase on magnetic particles through gene fusion.
Recombinant DNA technology has been utilized to fuse an octapeptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG), to the C-terminus of organophosphorus hydrolase (OPH, EC 3.1.8.1), an enzyme capable of hydrolyzing organophosphate compounds, such as insecticides and nerve gas agents. The recombinant OPH-FLAG was immobilized onto magnetic beads coated with protein A in the following ways: (a) site-directly through a monoclonal antibody (MAb) specific for the FLAG peptide; (b) through the MAb that was randomly tethered to the beads using glutaraldehyde; (c) randomly by cross-linking OPH-FLAG to protein-coated beads using glutaraldehyde. Kinetic studies demonstrated that the site-directly immobilized enzyme maintained the highest catalytic efficiency. The orientation specific immobilization strategy described in this article can be applied to other proteins, and therefore, it may find potential applications in the design of biosensors, biocatalysts, and bioreactors having immobilized proteins as their biorecognition elements.
Wang
J
J
Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506, USA.
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D000818:Animals; D000911:Antibodies, Monoclonal; D019677:Artificial Gene Fusion; D043303:Aryldialkylphosphatase; D004274:DNA, Recombinant / Q000235:genetics; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008280:Magnetics; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D004800:Enzymes, Immobilized; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C085706:FLAG peptide; D004950:Esterases; D043303:Aryldialkylphosphatase