title: Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E. coli and the baculovirus expression systems.
abstract: All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST). High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E. coli. The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg). NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (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 digestion with Factor Xa protease. Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient 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 immobilized 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.
authors.affiliation: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, QLD, Australia. A.Khromykh@mailbox.uq.oz.au
authors.forename: A A
authors.initials: AA
authors.lastname: Khromykh
authors.forename: T J
authors.initials: TJ
authors.lastname: Harvey
authors.forename: M
authors.initials: M
authors.lastname: Abedinia
authors.forename: E G
authors.initials: EG
authors.lastname: Westaway
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D005416:Flavivirus / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases; D018411:Spodoptera / Q000166:cytology; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D005982:Glutathione Transferase; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C100315:NS1 protein, Flavivirus; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080107:NS4B protein, flavivirus; C096041:NS5 protein, flavivirus