The cDNA template encoding the kinase domain of GNE was codon optimized for overexpression in E. coli and synthesized commercially (Codon Devices, Inc.) The DNA fragment encoding GNE residues 406-720 was PCR amplified and subcloned into the pET28-MHL vector (gi:134105571) using an In-Fusion dry-down PCR cloning kit (ClonTech). Protein was overexpressed in E. coli BL21(DE3) CodonPlus-RIL cells (Stratagene) grown in terrific broth medium. The culture was grown at 37uC in a LEX bubbling system (Harbinger Biotech. & Engineering Corp.) until OD 600 reached 3.0. The temperature of the culture was then lowered to 15uC and the cells were induced with 0.5 mM isopropyl 1-thio-b-D-galactopyranoside and allowed to grow further overnight. Cells were harvested by centrifugation and flash frozen in liquid nitrogen and stored at 280uC until purification. Frozen cells were thawed and resuspended in 10 mM HEPES buffer (pH 7.5) containing 500 mM sodium chloride, 5% glycerol, 2 mM b-mercaptoethanol, and supplemented with 5 mM imidazole, and mechanically lysed using a microfluidizer (Microfluidics, model M-110EH) at 1,000 bar pressure. The lysate was clarified by centrifugation. GNE protein was bound with nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) at a ratio of 2.5 mL 50% Ni-NTA flurry per litre of cell culture. The bound protein was washed twice with the same HEPES buffer containing 30 mM or 75 mM imidazole, and finally eluted with the HEPES buffer supplemented with 300 mM imidazole. The elutant containing the GNE protein was further purified by Supderdex-75 size exclusion chromatography (GE Healthcare). The eluted fractions were pooled, concentrated to a final concentration of 40 mg per mL, and stored in a buffer containing 10 mM HEPES, pH 7.5, 500 mM sodium chloride, 5% glycerol and 5 mM dithiothreitol. The purity of the protein was better than 95% judging from SDS-PAGE gel.