Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ

[Image: see text] Diacylglycerol kinase ζ (DGKζ) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGKζ has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGKζ, together with DGKα, as a potential target for cancer immunotherapy. However, DGKζ has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGKζ using a baculovirus–insect cell expression system for enzymological and structural studies. Full-length DGKζ remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGKζ is in a fully functional state. The K(m) values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGKα and DGKε, and its affinities for DG and phosphatidylserine are comparable to those of DGKα/DGKε. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGKζ is comprised of 25% α-helices and 18% β-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGKζ. The purification of DGKζ allows detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.