Expression of Oligohistidine-Tagged Ricin B Chain in Spodoptera frugiperda

Lawrence B. Afrin, Heather Gulick, Joseph Vesely, Mark Willingham, Arthur E. Frankel

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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 107 infectious particles/mL. Sf9 cells grown in serum-free medium were then infected at an moi of 3 in the presence of 25 mM α-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 Ni2+-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. The recombinant reassociated heterodimer not only demonstrated cytotoxicity to HPB–MLT human leukemia cells (ID50 10−12M) similar to ricin and reassociated plant RTA–plant RTB but also bound Ni2+–NTA resin, suggesting preservation of function of RTA, RTB, and the new ligand fused to RTB. Thus, the recombinant fusion of new ligands to RTB may represent a novel and practical method for developing new immunotoxins.

Original languageEnglish (US)
Pages (from-to)539-546
Number of pages8
JournalBioconjugate Chemistry
Volume5
Issue number6
DOIs
StatePublished - Nov 1 1994

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