Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).
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.
Azevedo
Cristina
C
Medical Research Council Cell Biology Unit and Laboratory for Molecular Cell Biology, Department of Cell Biology, University College London, London, UK.
Burton
Adam
A
Bennett
Matthew
M
Onnebo
Sara Maria Nancy
SM
Saiardi
Adolfo
A
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D007295:Inositol Phosphates / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007295:Inositol Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C402123:inositol heptakisphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C099438:inositol hexakisphosphate kinase