Characterization of single site ricin toxin B chain mutants

Arthur Frankel, Edward Tagge, John Chandler, Chris Burbage, Greg Hancock, Joseph Vesely, Mark Willingham

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Abbreviations: RTA. ricin toxin A chain; RTB. ricin toxin B chain; SPR, surface plasmon resonance DNA encoding ricin B chain was modified by site-directed mutagenesis, and eight separate mutant RTB cDNAs including four novel mutants were ligated into the baculovirus transfer vector, pAcGP67A. Cotransfection of S. frugiperda Sf9 cells with BaculoGold DNA was followed by limiting dilution isolation of recombinant baculoviruses. Infection of Sf9 cells at a multiplicity of infection of 5 in the presence of 25 mM lactose produced 0.05-1 mg/L of soluble, glycosylated 34 kDa proteins immunoreactive with monoclonal and polyclonal antibodies to ricin B chain. Mutant ricin B chains were partially purified by monoclonal antibody immunoaffinity chromatography to 10-50% purity in near milligram quantities. The mutant ricin B chains had decreased lectin binding relative to plant ricin B chain as measured by binding to immobilized lactose and asialofetuin and cell binding immunofluorescence. The mutant ricin B chains reassociated with plant RTA similarly to plant RTB, and the recombinant heterodimers had slightly reduced cell cytotoxicity relative to ricin.

Original languageEnglish (US)
Pages (from-to)30-37
Number of pages8
JournalBioconjugate Chemistry
Volume7
Issue number1
StatePublished - 1996

Fingerprint

Ricin
Rapid thermal annealing
DNA
Mutagenesis
Monoclonal antibodies
Surface plasmon resonance
Cytotoxicity
Chromatography
Antibodies
Dilution
Proteins
Sf9 Cells
Baculoviridae
Lactose
Monoclonal Antibodies
Surface Plasmon Resonance
Site-Directed Mutagenesis
Infection
Lectins
Fluorescent Antibody Technique

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Clinical Biochemistry
  • Chemistry(all)
  • Organic Chemistry

Cite this

Frankel, A., Tagge, E., Chandler, J., Burbage, C., Hancock, G., Vesely, J., & Willingham, M. (1996). Characterization of single site ricin toxin B chain mutants. Bioconjugate Chemistry, 7(1), 30-37.

Characterization of single site ricin toxin B chain mutants. / Frankel, Arthur; Tagge, Edward; Chandler, John; Burbage, Chris; Hancock, Greg; Vesely, Joseph; Willingham, Mark.

In: Bioconjugate Chemistry, Vol. 7, No. 1, 1996, p. 30-37.

Research output: Contribution to journalArticle

Frankel, A, Tagge, E, Chandler, J, Burbage, C, Hancock, G, Vesely, J & Willingham, M 1996, 'Characterization of single site ricin toxin B chain mutants', Bioconjugate Chemistry, vol. 7, no. 1, pp. 30-37.
Frankel A, Tagge E, Chandler J, Burbage C, Hancock G, Vesely J et al. Characterization of single site ricin toxin B chain mutants. Bioconjugate Chemistry. 1996;7(1):30-37.
Frankel, Arthur ; Tagge, Edward ; Chandler, John ; Burbage, Chris ; Hancock, Greg ; Vesely, Joseph ; Willingham, Mark. / Characterization of single site ricin toxin B chain mutants. In: Bioconjugate Chemistry. 1996 ; Vol. 7, No. 1. pp. 30-37.
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