RESUMO
The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.
Assuntos
Fluorometria/métodos , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Baculoviridae/genética , Domínio Catalítico , Linhagem Celular , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citologia , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.
