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

doi:10.1021/acs.analchem.5b00117

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

Biosciences

Research output: Contribution to journal › Article › Scientific › peer-review

9 Citations (Scopus)

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.

Original language	Undefined/Unknown
Pages (from-to)	3527–3534
Number of pages	8
Journal	Analytical Chemistry
Volume	87
Issue number	6
DOIs	https://doi.org/10.1021/acs.analchem.5b00117
Publication status	Published - 2015
MoE publication type	A1 Journal article-refereed

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10.1021/acs.analchem.5b00117

Cite this

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title = "GTP-Specific Fab Fragment-Based GTPase Activity Assay",

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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doi = "10.1021/acs.analchem.5b00117",

language = "Odefinierat/ok{\"a}nt",

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pages = "3527–3534",

journal = "Analytical Chemistry",

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T1 - GTP-Specific Fab Fragment-Based GTPase Activity Assay

AU - Kopra, K

AU - Rozwandowicz-Jansen, A

AU - Syrjänpää, M

AU - Blazevits, Olga

AU - Ligabue, Alessio

AU - Veltel, S

AU - Lamminmäki, U

AU - Abankwa, Daniel

AU - Härmä, H

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

U2 - 10.1021/acs.analchem.5b00117

DO - 10.1021/acs.analchem.5b00117

M3 - Artikel

SN - 0003-2700

VL - 87

SP - 3527

EP - 3534

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 6

ER -