Double-site ricin B chain mutants retain galactose binding

Arthur Frankel, Edward Tagge, John Chandler, Chris Burbage, Mark Willingham

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Three distinct double-site and two single-site ricin B chain (RTB) mutants were expressed in Spodoptera frugiperda insect cells and purified from infected cell supernatants. The yields of recombinant proteins were 0.01-0.2 mg/l. The purity after monoclonal antibody affinity chromatography was 1-20%. The mutant proteins were soluble, immunoreactive with monoclonal antibodies and polyclonal antibodies to RTB and demonstrated molecular weights of 32 kDa, similar to plant RTB. All three double-site and both single-site mutants bound asialofetuin and mammalian cell surfaces based on an asialofetuin ELISA and cell binding immunofluorescence assay. While one double-site mutant, W37S/Y248S, had a 1 log drop in sugar binding, the other two double-site mutants W37S/Y248H and D22E/D234E had 2 log reductions in sugar binding. Each mutant reassociated efficiently (25-75%) with plant ricin A chain (RTA) to form cytotoxic heterodimers. The concentration of protein required to reduce protein synthesis 50% (ID50) was 1 log higher than plant ricin for W37S/Y248S-RTA and the single-site mutant heterodimers, Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin for the other two double-site mutant heterodimers. The results suggest amino acid residues in both the 1α and 2γ subdomains of RTB participate in sugar binding. However, other subdomains must contribute to the avidity of ricin for cell surface oligosaccharides.

Original languageEnglish (US)
Pages (from-to)371-379
Number of pages9
JournalProtein Engineering
Volume9
Issue number4
Publication statusPublished - Apr 1996

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Keywords

  • Galactose binding
  • Lectin
  • Ricin

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Frankel, A., Tagge, E., Chandler, J., Burbage, C., & Willingham, M. (1996). Double-site ricin B chain mutants retain galactose binding. Protein Engineering, 9(4), 371-379.