Aa. Khromykh et al., EXPRESSION AND PURIFICATION OF THE 7 NONSTRUCTURAL PROTEINS OF THE FLAVIVIRUS KUNJIN IN THE ESCHERICHIA-COLI AND THE BACULOVIRUS EXPRESSIONSYSTEMS, Journal of virological methods, 61(1-2), 1996, pp. 47-58
Citations number
42
Categorie Soggetti
Virology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
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.