GTP-specific fab fragment-based GTPase activity assay

  • Anal Chem. 2015 Mar 17;87(6):3527-34. doi: 10.1021/acs.analchem.5b00117.
Kari Kopra  1 ,  Anita Rozwandowicz-Jansen ,  Markku Syrjänpää ,  Olga Blaževitš  2 ,  Alessio Ligabue  2 ,  Stefan Veltel  3 ,  Urpo Lamminmäki ,  Daniel Abankwa  2 ,  Harri Härmä  1
Affiliations
  • 1. †Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, Third Floor, FI-20520 Turku, Finland.
  • 2. ∥Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
  • 3. ⊥University Hospital Hamburg-Eppendorf, Hamburg, Germany.
PMID: 25707436 DOI: 10.1021/acs.analchem.5b00117
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.

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