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GTP-specific fab fragment-based GTPase activity assay

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GTP-specific fab fragment-based GTPase activity assay. / Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri.

In: ANAL CHEM, Vol. 87, No. 6, 17.03.2015, p. 3527-34.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Kopra, K, Rozwandowicz-Jansen, A, Syrjänpää, M, Blaževitš, O, Ligabue, A, Veltel, S, Lamminmäki, U, Abankwa, D & Härmä, H 2015, 'GTP-specific fab fragment-based GTPase activity assay', ANAL CHEM, vol. 87, no. 6, pp. 3527-34. https://doi.org/10.1021/acs.analchem.5b00117

APA

Kopra, K., Rozwandowicz-Jansen, A., Syrjänpää, M., Blaževitš, O., Ligabue, A., Veltel, S., Lamminmäki, U., Abankwa, D., & Härmä, H. (2015). GTP-specific fab fragment-based GTPase activity assay. ANAL CHEM, 87(6), 3527-34. https://doi.org/10.1021/acs.analchem.5b00117

Vancouver

Kopra K, Rozwandowicz-Jansen A, Syrjänpää M, Blaževitš O, Ligabue A, Veltel S et al. GTP-specific fab fragment-based GTPase activity assay. ANAL CHEM. 2015 Mar 17;87(6):3527-34. https://doi.org/10.1021/acs.analchem.5b00117

Bibtex

@article{0e41cb70b0e64820aa396dca3d4b7bcd,
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.",
author = "Kari Kopra and Anita Rozwandowicz-Jansen and Markku Syrj{\"a}np{\"a}{\"a} and Olga Bla{\v z}evit{\v s} and Alessio Ligabue and Stefan Veltel and Urpo Lamminm{\"a}ki and Daniel Abankwa and Harri H{\"a}rm{\"a}",
year = "2015",
month = mar,
day = "17",
doi = "10.1021/acs.analchem.5b00117",
language = "English",
volume = "87",
pages = "3527--34",
journal = "ANAL CHEM",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "6",

}

RIS

TY - JOUR

T1 - GTP-specific fab fragment-based GTPase activity assay

AU - Kopra, Kari

AU - Rozwandowicz-Jansen, Anita

AU - Syrjänpää, Markku

AU - Blaževitš, Olga

AU - Ligabue, Alessio

AU - Veltel, Stefan

AU - Lamminmäki, Urpo

AU - Abankwa, Daniel

AU - Härmä, Harri

PY - 2015/3/17

Y1 - 2015/3/17

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 - SCORING: Journal article

C2 - 25707436

VL - 87

SP - 3527

EP - 3534

JO - ANAL CHEM

JF - ANAL CHEM

SN - 0003-2700

IS - 6

ER -