Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).

Methods in Molecular Biology
Cristina AzevedoAdolfo Saiardi

Abstract

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.

Citations

Jan 12, 2011·Proceedings of the National Academy of Sciences of the United States of America·David MaagAdam C Resnick
Jul 22, 2011·The Journal of Biological Chemistry·Annalisa LonettiAdolfo Saiardi
Sep 14, 2011·Journal of Visualized Experiments : JoVE·Omar LossAdolfo Saiardi
Dec 20, 2017·Nature Communications·Doreen BlüherUlla Bonas
Feb 23, 2013·Pharmacological Reviews·Christopher J Barker, Per-Olof Berggren
Nov 23, 2019·Proceedings of the National Academy of Sciences of the United States of America·Yann DesfougèresAdolfo Saiardi
Mar 23, 2017·Proceedings of the National Academy of Sciences of the United States of America·Eun Suk SongLouis B Hersh
Aug 29, 2009·Cellular and Molecular Life Sciences : CMLS·Christopher John BarkerPer-Olof Berggren
Feb 6, 2017·Proceedings of the National Academy of Sciences of the United States of America·Chenglai FuSolomon H Snyder

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