Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking

Edward Tagge, Billie Harris, Chris Burbage, Philip Hall, Joseph Vesely, Mark Willingham, Arthur Frankel

Research output: Contribution to journalArticle

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Abstract

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 °C and, on incubation at 37 °C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgi-dependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

Original languageEnglish (US)
Pages (from-to)743-750
Number of pages8
JournalBioconjugate Chemistry
Volume8
Issue number5
DOIs
StatePublished - Sep 1997

Fingerprint

Ricin
Green Fluorescent Proteins
Proteins
Monitoring
Fusion reactions
KB Cells
Rapid thermal annealing
Fluorescence
Cells
Genetic engineering
Galactosides
Genetic Engineering
Escherichia coli Proteins
Endosomes
Electric fuses
enhanced green fluorescent protein
Escherichia coli
Microscopy
Hepatocellular Carcinoma
Squamous Cell Carcinoma

ASJC Scopus subject areas

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

Cite this

Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking. / Tagge, Edward; Harris, Billie; Burbage, Chris; Hall, Philip; Vesely, Joseph; Willingham, Mark; Frankel, Arthur.

In: Bioconjugate Chemistry, Vol. 8, No. 5, 09.1997, p. 743-750.

Research output: Contribution to journalArticle

Tagge, E, Harris, B, Burbage, C, Hall, P, Vesely, J, Willingham, M & Frankel, A 1997, 'Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking', Bioconjugate Chemistry, vol. 8, no. 5, pp. 743-750. https://doi.org/10.1021/bc9700749
Tagge, Edward ; Harris, Billie ; Burbage, Chris ; Hall, Philip ; Vesely, Joseph ; Willingham, Mark ; Frankel, Arthur. / Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking. In: Bioconjugate Chemistry. 1997 ; Vol. 8, No. 5. pp. 743-750.
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