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

D. Fryxell, B. Y. Li, D. Mohanraj, B. Johnson, S. Ramakrishnan

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)253-259
Number of pages7
JournalBiochemical and biophysical research communications
Volume210
Issue number2
DOIs
StatePublished - May 16 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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