title: Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain.
abstract: Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: A E
authors.initials: AE
authors.lastname: Shannon
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Chappell
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: M J
authors.initials: MJ
authors.lastname: Stoermer
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: S Y
authors.initials: SY
authors.lastname: Chow
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: W M
authors.initials: WM
authors.lastname: Kok
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: D P
authors.initials: DP
authors.lastname: Fairlie
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia; Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia. Electronic address: p.young@uq.edu.au.
authors.forename: P R
authors.initials: PR
authors.lastname: Young
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D003716:Dengue Virus / Q000201:enzymology*; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011480:Protease Inhibitors / Q000737:chemistry; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases
keywords: Cofactor inhibitor; Dengue virus; NS2B–NS3; Protease