GTP-Specific Fab Fragment-Based GTPase Activity Assay

K Kopra, A Rozwandowicz-Jansen, M Syrjänpää, Olga Blazevits, Alessio Ligabue, S Veltel, U Lamminmäki, Daniel Abankwa, H Härmä

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6 Citeringar (Scopus)

Sammanfattning

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.
OriginalspråkOdefinierat/okänt
Sidor (från-till)3527–3534
Antal sidor8
TidskriftAnalytical Chemistry
Volym87
Utgåva6
DOI
StatusPublicerad - 2015
MoE-publikationstypA1 Tidskriftsartikel-refererad

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