A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin

Analytical Biochemistry
Diana V Pastrana, D J FitzGerald

Abstract

Pseudomonas exotoxin A (PE) inhibits protein synthesis by NAD-dependent ADP-ribosylation of eukaryotic elongation factor 2. Traditionally, toxin activity has been characterized, either in living cells or cell-free systems, using radioactive compounds for quantification. The increased costs of radioactive waste disposal together with heightened security concerns have made the use of radioactive isotopes less attractive for routine laboratory assays. We therefore adapted a cell-free rabbit reticulocyte in vitro transcription-translation system that utilizes a reporter (beta-galactosidase) to measure toxin activity. The assay for PE is rapid, scalable, log-linear, NAD dependent and can be used to assess the neutralizing activity of anti-PE antibody preparations.

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Citations

Nov 22, 2011·Protein Engineering, Design & Selection : PEDS·Wenhai LiuIra Pastan
Jan 14, 2009·Applied and Environmental Microbiology·Beatriz QuiñonesKen Teter
Sep 29, 2009·Biologicals : Journal of the International Association of Biological Standardization·Yunpeng SuArthur E Frankel
Jun 20, 2018·ACS Chemical Biology·Thomas PirzerHarald Kolmar
Oct 1, 2020·Bioconjugate Chemistry·Audrey StoesselMariel Donzeau

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