Recombinant Enzyme Purification and Mutagenesis

Recombinant enzyme was prepared essentially as described previously [12 (link)]. Protein from PIP4K2C (UniGene 6280511) or associated mutants, cloned into the expression vector pGEX6P (GE Healthcare) was expressed and purified from Escherichia coli BL21(DE3). Cultures were induced with 0.4 mM IPTG and probe-sonicated in the presence of protease inhibitors. GST fusion proteins of PI5P4Kγ and PI5P4Kγ+, a mutant with specific activity close to that of the active PI5P4Kα isoform [12 (link)], were harvested by binding to glutathione sepharose beads (GE Healthcare) and cleaved in situ with 50 units of PreScission protease (GE Healthcare) for 4 h at 4°C. Purity was confirmed by SDS/PAGE and protein concentration determined by colorimetric assay (Bio-Rad). Site-directed mutagenesis using the QuikChange technique (Agilent Technologies) was used to generate clones from which mutant forms of PI5P4Kγ and PI5P4Kγ+ were produced (for mutagenesis primers see Supplementary Table S1).