Genetic construction of a phosphorylation site in ricin a chain: Specific radiolabeling of recombinant proteins for localization and degradation studies

Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-³²P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The ³²P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [³²P] phosphorylation.