Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1)
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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-IP5 or IP7) 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 5β[32P] InsP 7 that we subsequently use in vitro experiments to phosphorylate proteins extracts from different species.
Key words:Inositol Pyrophosphate IP6K1 Phosphorylation Protein purification Enzymatic reaction
This work is supported by MRC funding of the Cell Biology Unit and by EC through an IRG (014827).