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Old 11-19-2010, 08:44 PM
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Default Design, high-level expression, purification and characterization of soluble fragments

Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

Related Articles Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

Protein Eng. 2001 Aug;14(8):573-82

Authors: Gesell JJ, Liu D, Madison VS, Hesson T, Wang YS, Weber PC, Wyss DF

RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.

PMID: 11579226 [PubMed - indexed for MEDLINE]



Source: PubMed
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