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 tag 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 [gamma-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.
Fryxell
D
D
Department of Pharmacology, University of Minnesota, Minneapolis, USA.
Li
B Y
BY
Mohanraj
D
D
Johnson
B
B
Ramakrishnan
S
S
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012276:Ricin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D009842:Oligopeptides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D006639:Histidine; C037642:kemptide; D012276:Ricin