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GTP-specific fab fragment-based GTPase activity assay

Kari Kopra1, Anita Rozwandowicz-Jansen, Markku Syrjänpää

  • 1†Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, Third Floor, FI-20520 Turku, Finland.

Analytical Chemistry
|February 25, 2015
PubMed

Insights

Researchers developed a novel GTP-specific antibody fragment to detect free guanosine triphosphate (GTP). This tool enables sensitive monitoring of GTP levels and GTPase activity in cellular signaling pathways.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • GTPases are crucial for cellular signaling, cycling between inactive (GDP-bound) and active (GTP-bound) states.
  • Ras GTPases are frequently altered in cancer, yet effective inhibitors are limited.
  • Current methods for monitoring GTP hydrolysis often use luminescent analogs.

Purpose of the Study:

  • To develop and characterize a novel GTP-specific antibody fragment.
  • To create a sensitive assay for monitoring GTP concentration and GTPase activity.

Main Methods:

  • Phage display technology was used to select antibody fragments against GTP-biotin conjugates.
  • The most potent fragment, 2A4(GTP), was identified and characterized for its specificity.
  • A heterogeneous time-resolved luminescence assay was developed using the 2A4(GTP) fragment.

Main Results:

  • Six Fab fragments with specificity for GTP were selected.
  • The 2A4(GTP) fragment demonstrated over 100-fold specificity for GTP compared to GDP, ATP, or CTP.
  • The developed assay can monitor GTP concentration and GTPase activity (H-Ras activation/deactivation) at nanomolar protein levels.

Conclusions:

  • A novel, highly specific GTP-binding Fab fragment (2A4(GTP)) was successfully developed.
  • This fragment enables a sensitive luminescence-based assay for studying GTPase function.
  • The assay facilitates research into cellular signaling pathways involving GTPases, particularly Ras proteins.

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