EXPRESSION OF OLIGOHISTIDINE-TAGGED RICIN-B CHAIN IN SPODOPTERA-FRUGIPERDA

DNA encoding ADPGH(6)G was fused to the 5'-end of RTB DNA and subclone d as a BamHI-EcoRI DNA cassette into the baculovirus transfer vector, pAcGP67A. Spodoptera frugiperda Sf9 cells were cotransfected with pAcG P67A-ADPGH(6)G-RTB DNA and BaculoGold AcNPV DNA, and recombinant bacul ovirus was isolated by two cycles of limiting dilution assay followed by dot blot analysis with P-32-dCTP random primer labeled RTB DNA. Rec ombinant virus was purified and amplified to obtain stocks at titers o f 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-lacto se. After 5 days, supernatants and cell pellets were harvested and ass ayed by an asialofetuin ELISA for recombinant RTB protein. Fusion RTB protein was produced in the supernatant at 5 mg/L and in the cell pell et at 1 mg/L. Recombinant protein was purified to >80% homogeneity usi ng either a monoclonal antibody affinity matrix with alkaline elution or a Ni2+-NTA matrix with imidazole elution. The purified protein boun d asialofetuin similarly to plant RTB. N-terminal sequencing confirmed the oligohistidine tag. SDS-PAGE confirmed the 1,000 Da increase in m ass relative to ''wild-type'' recombinant RTB produced in Sig cells. I mmunoblots confirmed reactivity with polyclonal and monoclonal antibod ies to plant RTB. The fusion protein reassociated with plant RTA simil arly to plant RTE. The recombinant reassociated heterodimer not only d emonstrated cytotoxicity to HPB-MLT human leukemia cells (ID50 10(-12) M) similar to ricin and reassociated plant RTA-plant RTB but also boun d Ni2+-NTA resin, suggesting preservation of function of RTA, RTB, and the new ligand fused to RTB. Thus, the recombinant fusion of new liga nds to RTB may represent a novel and practical method for developing n ew immunotoxins.