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

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

SUBMITTER: Takahashi D 

PROVIDER: S-EPMC6089211 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature