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 mutageni zed toxin was as demonstrated by labeling with [gamma-P-32] ATP. Kempt ide-A chain could be labeled even after reassociation with ricin B cha in or disulfide linkage to antibody to form an immunotoxin. The P-32 l abel in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Ke mptide-immunotoxin was tested in cytotoxicity assays and used to monit or internalization of the toxin moiety after [P-32] phosphorylation. ( C) 1995 Academic Press, Inc.