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Biotechnol Bioeng ; 95(1): 48-57, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16673413

RESUMO

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.


Assuntos
Cromatografia de Afinidade/métodos , Cobalto/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/isolamento & purificação , Inativação de Vírus/efeitos dos fármacos , Cobalto/química , Radicais Livres/farmacologia , Vetores Genéticos/efeitos dos fármacos , Vetores Genéticos/isolamento & purificação , Herpesvirus Humano 1/genética , Radical Hidroxila/farmacologia , Estresse Oxidativo/genética , Manejo de Espécimes/métodos
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