RESUMO
Hepatitis C Virus c33, a recombinant protein comprising residues 1192-1457 of NS3 helicase, has been a mainstay of HCV serology for decades. With seven unpaired cysteines, seroreactivity of E. coli expressed c33 is dependant on reductants. While engineering a c33 replacement for new anti-HCV serological tests, we sought to reduce oxidation sensitivity, a liability for immunodiagnostic reagent stability. A series of cysteine-to-serine substituted variants of a c33-like antigen was constructed and evaluated for reactivity against a panel of HCV-positive sera. Several variants were essentially nonreactive while others exhibited reactivity similar to or better than the wild-type construct. One demonstrated equivalent potency to wild-type but also diminished DTT dependence. To explore enhanced anti-NS3 reactivity, we constructed and examined an expanded series of antigens comprising individual helicase domains, the full-length helicase, additional cysteine-to-serine variants, and variants at positions critical to catalytic activity. Immunoassays using these latter NS3 helicase recombinants demonstrated that domain 1 possessed significantly more seroreactivity than previously believed, that the use of soluble full-length helicase protein enhanced sensitivity by several-fold over c33, and that anti-NS3 helicase seroreactivity was further enhanced by the introduction of point mutations which altered the catalytic activity or oxidation sensitivity of the antigen.
Assuntos
DNA Helicases/genética , DNA Helicases/imunologia , Hepacivirus/enzimologia , Hepacivirus/genética , Testes Sorológicos , Proteínas não Estruturais Virais/genética , Anticorpos Antivirais/sangue , Cisteína/genética , Cisteína/imunologia , DNA Helicases/metabolismo , Escherichia coli/genética , Engenharia Genética , Hepacivirus/imunologia , Humanos , Testes Imunológicos , Mutação Puntual , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Soroconversão , Proteínas não Estruturais Virais/imunologiaRESUMO
Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise.
