Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH

Mathewos Tessema, Peter C. Simons, Daniel F. Cimino, Lilliana Sanchez, Anna Waller, Richard G Posner, Angela Wandinger-Ness, Eric R. Prossnitz, Larry A. Sklar

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Background: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. Methods: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. Results: A 10 γl assay can measure 5 μl of 20 μM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. Conclusions: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.

Original languageEnglish (US)
Pages (from-to)326-334
Number of pages9
JournalCytometry Part A
Volume69
Issue number5
DOIs
StatePublished - May 2006

Fingerprint

Green Fluorescent Proteins
Glutathione Transferase
Glutathione
Proteins
Flow Cytometry
Affinity Chromatography
Protein Binding
Glutathione Disulfide
Protein S
antineoplaston A10
Dextrans
Oxidation-Reduction
Carrier Proteins
Buffers
Western Blotting

Keywords

  • Glutathione assay
  • GST fusion protein
  • GST pulldown
  • GST-GFP

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH. / Tessema, Mathewos; Simons, Peter C.; Cimino, Daniel F.; Sanchez, Lilliana; Waller, Anna; Posner, Richard G; Wandinger-Ness, Angela; Prossnitz, Eric R.; Sklar, Larry A.

In: Cytometry Part A, Vol. 69, No. 5, 05.2006, p. 326-334.

Research output: Contribution to journalArticle

Tessema, M, Simons, PC, Cimino, DF, Sanchez, L, Waller, A, Posner, RG, Wandinger-Ness, A, Prossnitz, ER & Sklar, LA 2006, 'Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH', Cytometry Part A, vol. 69, no. 5, pp. 326-334. https://doi.org/10.1002/cyto.a.20259
Tessema, Mathewos ; Simons, Peter C. ; Cimino, Daniel F. ; Sanchez, Lilliana ; Waller, Anna ; Posner, Richard G ; Wandinger-Ness, Angela ; Prossnitz, Eric R. ; Sklar, Larry A. / Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH. In: Cytometry Part A. 2006 ; Vol. 69, No. 5. pp. 326-334.
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AU - Cimino, Daniel F.

AU - Sanchez, Lilliana

AU - Waller, Anna

AU - Posner, Richard G

AU - Wandinger-Ness, Angela

AU - Prossnitz, Eric R.

AU - Sklar, Larry A.

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