Characterization of single site ricin toxin B chain mutants.
DNA encoding ricin B chain was modified by site-directed mutagenesis, and eight separate mutant RTB cDNAs including four novel mutants were ligated into the baculovirus transfer vector, pAcGP67A. Cotransfection of S. frugiperda Sf9 cells with BaculoGold DNA was followed by limiting dilution isolation of recombinant baculoviruses. Infection of Sf9 cells at a multiplicity of infection of 5 in the presence of 25 mM lactose produced 0.05-1 mg/L of soluble, glycosylated 34 kDa proteins immunoreactive with monoclonal and polyclonal antibodies to ricin B chain. Mutant ricin B chains were partially purified by monoclonal antibody immunoaffinity chromatography to 10-50% purity in near milligram quantities. The mutant ricin B chains had decreased lectin binding relative to plant ricin B chain as measured by binding to immobilized lactose and asialofetuin and cell binding immunofluorescence. The mutant ricin B chains reassociated with plant RTA similarly to plant RTB, and the recombinant heterodimers had slightly reduced cell cytotoxicity relative to ricin.
Frankel
A
A
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Tagge
E
E
Chandler
J
J
Burbage
C
C
Hancock
G
G
Vesely
J
J
Willingham
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae; D001665:Binding Sites; D002470:Cell Survival / Q000187:drug effects; D018076:DNA, Complementary; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007624:KB Cells; D037102:Lectins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D012276:Ricin / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D018411:Spodoptera; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D037102:Lectins; D011994:Recombinant Proteins; D012276:Ricin