Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies

摘要: Diacylglycerol kinases (DGKs) are lipid that modulate the levels of second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. DGKα consists N-terminal regulatory domains including EF-hand motifs C1 domains, C-terminal catalytic domain (DGKα-CD). To date, however, no structures mammalian DGKs their CDs have yet reported, impeding our understanding on mechanism rational structure-based drug design. Here we attempted produce DGKα-CD or full-length using bacterial baculovirus-insect cell expression system structural studies. While several constructs produced both insect cells formed insoluble soluble aggregates, expressed in remained was purified near homogeneity monomer with yields (1.3 mg/mL per one L culture) feasible protein crystallization. Following enzymatic characterization showed is fully functional state. We further demonstrated enzyme could be concentrated without any significant aggregation, characterized secondary structure by circular dichroism. Taken together, these results suggest presence suppress aggregation likely via intramolecular interactions DGKα-CD, demonstrate form DGKα, not alone, represents promising approach sample studies DGKα. Thus, study will encourage future efforts determine crystal DGK, which determined since it first identified 1959.