Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence

Mary O'Hare, Alex N. Brown, Khalid Hussain, Angelika Gebhardt, Graham Watson, Lynne M. Roberts, Ellen S. Vitetta, Philip E. Thorpe, J. Michael Lord

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

Original languageEnglish (US)
Pages (from-to)200-204
Number of pages5
JournalFEBS Letters
Volume273
Issue number1-2
DOIs
StatePublished - Oct 29 1990

Keywords

  • Fusion protein
  • Protein A
  • Proteolytically-cleavable sequence
  • Ricin A chain

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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