Expression of oligohistidine-tagged ricin B chain in Spodoptera frugiperda.
DNA encoding ADPGH6G was fused to the 5'-end of RTB DNA and subcloned as a BamHI-EcoRI DNA cassette into the baculovirus transfer vector, pAcGP67A. Spodoptera frugiperda Sf9 cells were cotransfected with pAcGP67A-ADPGH6G-RTB DNA and BaculoGold AcNPV DNA, and recombinant baculovirus was isolated by two cycles of limiting dilution assay followed by dot blot analysis with 32P-dCTP random primer labeled RTB DNA. Recombinant virus was purified and amplified to obtain stocks at titers of 10(7) infectious particles/mL. Sf9 cells grown in serum-free medium were then infected at an moi of 3 in the presence of 25 mM alpha-lactose. After 5 days, supernatants and cell pellets were harvested and assayed by an asialofetuin ELISA for recombinant RTB protein. Fusion RTB protein was produced in the supernatant at 5 mg/L and in the cell pellet at 1 mg/L. Recombinant protein was purified to > 80% homogeneity using either a monoclonal antibody affinity matrix with alkaline elution or a Ni(2+)-NTA matrix with imidazole elution. The purified protein bound asialofetuin similarly to plant RTB. N-terminal sequencing confirmed the oligohistidine tag. SDS-PAGE confirmed the 1,000 Da increase in mass relative to "wild-type" recombinant RTB produced in Sf9 cells. Immunoblots confirmed reactivity with polyclonal and monoclonal antibodies to plant RTB. The fusion protein reassociated with plant RTA similarly to plant RTB.(ABSTRACT TRUNCATED AT 250 WORDS)
Afrin
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Department of Medicine, University of South Carolina, Charleston 29425.
Gulick
H
H
Vesely
J
J
Willingham
M
M
Frankel
A E
AE
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001212:Asialoglycoproteins / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D060748:Fetuins; D015870:Gene Expression; D006639:Histidine; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D012276:Ricin / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism
D000970:Antineoplastic Agents; D001212:Asialoglycoproteins; D060748:Fetuins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D006639:Histidine; D009532:Nickel; D012276:Ricin