Optimized expression and purification of adipose triglyceride lipase improved hydrolysis and transacylation catalytic activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E. coli, the insect cell line Sf9, and the mammalian cell line HEK 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1 to D288 flanked with N- and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of co-purified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared to previously published data, and it could be stimulated by the co-activator protein comparative gene identification 58 (CGI-58) and inhibited by the protein G0/G1 switch protein 2 (G0S2). Shock-freezing and storage did not affect the basal activity, but reduced co-activation of ATGL by CGI-58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as tri- and monoacylglycerol. However, the ATGL variant neither showed hydrolytic nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.