Mammalian Diacylglycerol Kinase Epsilon: Expression in Sf21 Cells, Purification, and Characterization

This is the first report of the purification and characterization of the arachidonoyl‐specific diacylglycerol kinase epsilon (DGKε). We have purified human DGKε and a truncated form lacking the first 40 residues (DGKεΔ40) to near homogeneity using Nickel‐affinity chromatography. Enzyme activity measurements showed that both purified constructs retained their substrate acyl chain specificity and have a specific activity comparable to N‐terminally FLAG epitope tagged forms of these proteins expressed in Cos‐7 cells. Thus, it is unlikely that the activity of DGKε is dependent on components present in cruder preparations. We observed that purified DGKε activity is lost rapidly and that freeze thawing is particularly damaging. We show that storage in 50% glycerol stabilizes DGKε significantly at 4°C, ‐80 °C and during freeze thawing. Secondary structure analyses using circular dichroism revealed that DGKε contains both α‐helical (~19%) and β‐structure (~25%), in agreement with predictive algorithms. DGKε exhibited a gradual and irreversible loss of secondary structure on heating to 100°C with slight stabilization upon adding dioleoyl‐phosphatidylcholine (DOPC). In contrast, DGKεΔ40 retained less structure at 100°C and showed a cooperative denaturation at about 80 o C with DOPC present. Interestingly, atomic force microscopy shows that DGKεΔ40 forms smaller and fewer aggregates on mica and on supported lipid bilayers (SLBs) compared to DGKε, despite both forms displaying similar activity trends. Aggregation on SLBs was reduced by the addition of a preferred substrate. This study provides the first successful purification of DGKε and a partial characterization of its enzymatic and conformational properties.