Baculovirus expression and characterization of catalytically active horseradish peroxidase.
Studies of horseradish peroxidase (HRP), a prototypical enzyme, have provided much of the information that is available on the mechanisms and functions of hemoprotein peroxidases. HRP itself is widely used in biotechnological applications. Further progress in defining the structure and function of the enzyme, however, requires its expression in a heterologous system. We report here baculovirus-mediated, high yield expression of a synthetic gene for HRP in Spodoptera frugiperda cell culture. Expression of the soluble, glycosylated protein requires the 5'-leader sequence of the native gene. Recombinant horseradish peroxidase reacts with H2O2 to give compound I, II, and III spectra and a guaiacol oxidation activity, identical to those of the native enzyme. The integrity of the recombinant active site is confirmed by NMR spectroscopy and by catalytic reaction with ethylhydrazine to give a stabilized isoporphyrin that decays exclusively to delta-meso-ethylheme. Furthermore, thioanisoles are oxidized by recombinant and native HRP with the same enantiomeric specificity. HRP expressed in a baculovirus system, despite probable differences in glycosylation, is essentially identical to the native enzyme.
Hartmann
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Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Ortiz de Montellano
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013379:Substrate Specificity; D014162:Transfection
D011994:Recombinant Proteins; D006735:Horseradish Peroxidase