Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography.
An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design.
Centre for Drug Design and Development, Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld. 4072, Australia.
Tracy L
TL
Arakaki
Ning-Xia
NX
Fang
David P
DP
Fairlie
Paul R
PR
Young
Jennifer L
JL
Martin
D002384:Catalysis; D002850:Chromatography, Gel; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D009097:Multienzyme Complexes; D020365:RNA Helicases; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D009097:Multienzyme Complexes; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases