基于GTP特异性Fab片段的GTP酶活性测定。

GTP-specific fab fragment-based GTPase activity assay.

作者信息

Kopra Kari, Rozwandowicz-Jansen Anita, Syrjänpää Markku, Blaževitš Olga, Ligabue Alessio, Veltel Stefan, Lamminmäki Urpo, Abankwa Daniel, Härmä Harri

机构信息

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

∥Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.

出版信息

Anal Chem. 2015 Mar 17;87(6):3527-34. doi: 10.1021/acs.analchem.5b00117. Epub 2015 Mar 3.

DOI:10.1021/acs.analchem.5b00117

PMID:25707436

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.

摘要

GTP酶是细胞信号传导的核心蛋白，它们以可控的方式在结合GDP的无活性构象和结合GTP的活性构象之间循环。Ras GTP酶在癌症中经常发生突变，到目前为止只有少数实验性抑制剂。监测GTP水解的最常用方法依赖于发光的GDP或GTP类似物。在本研究中，描述了首个GTP特异性Fab片段及其应用。我们使用噬菌体展示技术筛选Fab片段。发现了6个针对2'/3'-GTP-生物素和8-GTP-生物素的Fab片段。筛选出的抗体片段能够特异性检测内源性游离GTP。最有效的Fab片段（2A4(GTP)）对GTP的特异性比对GDP、ATP或CTP高100倍以上，并被用于开发一种基于异相时间分辨发光的检测方法来监测GTP浓度。该方法能够在纳摩尔蛋白浓度下研究GEF依赖的H-Ras激活（GTP结合）和GAP催化的H-Ras失活（GTP水解）。