2. Academic Validation
  3. GTP-specific fab fragment-based GTPase activity assay

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

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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