Azevedo Cristina, Burton Adam, Bennett Matthew, Onnebo Sara Maria Nancy, Saiardi Adolfo
Medical Research Council Cell Biology Unit and Laboratory for Molecular Cell Biology, Department of Cell Biology, University College London, London, UK.
Methods Mol Biol. 2010;645:73-85. doi: 10.1007/978-1-60327-175-2_5.
Soluble inositol polyphosphates represent a variegate class of signalling molecules essential for the function of disparate cellular processes. Recently, the phytic acid derivate inositol pyrophosphate, InsP(7) (PP-IP(5) or IP(7)) has been shown to pyro-phosphorylate proteins in a kinase independent way. To begin to understand the functional importance of this new phosphorylation mechanism, a source of cold and radiolabelled InsP(7) is indispensable. However, cold InsP(7) is expensive to buy, and labelled InsP(7) is not commercially available. Here we provide a protocol to synthesise and purify InsP(7) to a level of purity required for in vivo and in vitro experiments. We begin by purifying recombinant mouse inositol hexakisphosphate kinase (IP6K1) from Escherichia coli. With purified IP6K1, we produce cold InsP(7) and 5beta[(32)P] InsP(7) that we subsequently use in vitro experiments to phosphorylate proteins extracts from different species.
可溶性肌醇多磷酸是一类多样的信号分子，对不同细胞过程的功能至关重要。最近，植酸衍生物肌醇焦磷酸InsP(7)（PP-IP(5)或IP(7)）已被证明能以不依赖激酶的方式使蛋白质发生焦磷酸化。为了开始理解这种新磷酸化机制的功能重要性，冷的且带有放射性标记的InsP(7)来源是必不可少的。然而，冷的InsP(7)购买成本高昂，且带标记的InsP(7)没有商业供应。在此，我们提供了一种合成和纯化InsP(7)的方案，使其达到体内和体外实验所需的纯度水平。我们首先从大肠杆菌中纯化重组小鼠肌醇六磷酸激酶（IP6K1）。利用纯化的IP6K1，我们制备了冷的InsP(7)和5β[(32)P]InsP(7)，随后将其用于体外实验，使来自不同物种的蛋白质提取物发生磷酸化。
