Recombinant human IP6K2 and the human IPMK and PPIP5K2 kinase domain were prepared as previously described.41 (link),47 ,48 (link) Human IP3KA kinase domain (residues 173–461, UniProtKB code P23677) was cloned into the pDest-566 vector by the Gateway expression system (Invitrogen).48 (link) Genscript synthesized the codon-optimized cDNAs for expression in Escherichia coli of 6xHis-MBP tagged human IP6K1 (N-terminally tagged with 6×-His followed by MBP) and IP6K3 (N-terminally tagged with 6×-His followed by Sumo). The procedures for IP6K1, IP6K3, IPMK, and IP3KA expression and purification were identical to those used for IP6K2 except that the MBP tag was cut by TEV protease in IPMK and IP3KA. The cells were disrupted using a constant cell disruption system (Constant Systems) under 20 kpsi. Recombinant IP6Ks were purified with a Ni-NTA agarose column (Qiagen) followed by a HiTrap heparin HP column (GE Healthcare). As a final step, a Superdex 200 gel filtration column (GE Healthcare) was used with a running buffer of 150 mM NaCl and 20 mM Tris-HCl, pH 7.5. The purity of these proteins was estimated to be >80% as judged by SDS-PAGE. The purified proteins were stored in aliquots at −80 °C.