EXPRESSION AND PURIFICATION OF THE 7 NONSTRUCTURAL PROTEINS OF THE FLAVIVIRUS KUNJIN IN THE ESCHERICHIA-COLI AND THE BACULOVIRUS EXPRESSIONSYSTEMS

All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) r anging from NS1 to NS5 were expressed either alone or as fusion protei ns with Glutathione-S-transferase (GST). High level expression of reco mbinant proteins was achieved in Spodoptera frugiperda (Sf9) cells usi ng the baculovirus expression system in contrast to the low level of e xpression in E. coli. The order of the level of expression of the reco mbinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields s imilar to 4-5 mg) > GST-Delta NS3 (similar to 1-2 mg) > GST-4A (simila r to 1 mg) > GST-2B (similar to 0.5-1 mg) > GST-2A (similar to 0.5 mg) > GST-4B (similar to 0.1-0.2 mg). NS1 protein was expressed in a nati ve form at the level of similar to 2-4mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sep harose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E. coli cultures transformed with the expression plasmid and induced with IPTG. Only Delta NS3 protein was recovered intact by removing GST from the fusion protein by digest ion with Factor Xa protease. Attempts to cleave off the GST moiety fro m all the other purified recombinant proteins resulted either in ineff icient cleavage or in degradation of the proteins. No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-Delta NS3, GST-NS4A , and GST-NS4B was eluted off the GS beads by adding glutathione. Thus , KUN purified recombinant proteins, either in eluted form or while im mobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions.