Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography.
An engineered horseradish peroxidase isozyme C (HRP C) gene was constructed by the addition of a 6xArg fusion tail to 6xHis-HRP C by the PCR strategy. The 6xHis-6xArg-HRP C cDNA was expressed in the Sf9 insect cell line from Spodoptera frugiperda infected with Autographa californica nuclear polyhedrosis virus. The recombinant peroxidase isoelectric point was 9.5 as judged by isoelectric focusing and was purified directly from the culture medium at day-6 post-infection by cation-exchange chromatography or immobilised metal ion-affinity chromatography. While the former technique gave a yield of 98.5% with a purification factor of 130, the latter gave only a 68% yield with a purification factor of 140. Results obtained provide evidence that the poly-Arg tag is more effective than the poly-His tag for peroxidase purification from a baculovirus expression system.
Levin
Gustavo
G
Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica (UBA), Junín 956, 1113 Buenos Aires, Argentina.
Mendive
Fernando
F
Targovnik
Héctor M
HM
Cascone
Osvaldo
O
Miranda
María V
MV
D016367:Baculoviridae; D002852:Chromatography, Ion Exchange / Q000379:methods; D015870:Gene Expression; D006735:Horseradish Peroxidase / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D010455:Peptides; D011993:Recombinant Fusion Proteins; C015462:polyarginine; D006735:Horseradish Peroxidase