Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions.
Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.
Shivashankar
Y
Y
Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
Satchidanandam
V
V
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018349:Encephalitis Viruses, Japanese; D005982:Glutathione Transferase / Q000378:metabolism*; D008969:Molecular Sequence Data; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D005982:Glutathione Transferase; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases