GTP-Specific Fab Fragment-Based GTPase Activity Assay

A1 Journal article (refereed)


Internal Authors/Editors


Publication Details

List of Authors: Kopra K, Rozwandowicz-Jansen A, Syrjänpää M, Blazevits O, Ligabue A, Veltel S, Lamminmäki U, Abankwa D, Härmä H
Publisher: AMER CHEMICAL SOC
Publication year: 2015
Journal: Analytical Chemistry
Journal acronym: ANAL CHEM
Volume number: 87
Issue number: 6
Start page: 3527
End page: 3534
Number of pages: 8
ISSN: 0003-2700
eISSN: 1520-6882


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 2019-16-10 at 03:28

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