A1 Journal article – refereed
GTP-Specific Fab Fragment-Based GTPase Activity Assay




List of Authors: Kopra K, Rozwandowicz-Jansen A, Syrjanpaa M, Blazevits O, Ligabue A, Veltel S, Lamminmaki U, Abankwa D, Harma H
Publisher: AMER CHEMICAL SOC
Publication year: 2015
Journal: Analytical Chemistry
Journal name in source: ANALYTICAL CHEMISTRY
Journal acronym: ANAL CHEM
Volume number: 87
Issue number: 6
ISSN: 0003-2700

Abstract


GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.



Last updated on 2019-29-01 at 14:20