Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro
| Autor: | Renate Schreiber, Raymond J. Owens, Monika Oberer, Roland Viertlmayr, Natalia Kulminskaya, Christoph Heier, Mariana Colaço-Gaspar, Rudolf Zechner, Peter Hofer, Robert Zimmermann, Claudia Radler |
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| Rok vydání: | 2021 |
| Předmět: |
Acylation
Gene Expression Hormone-sensitive lipase PNPLA patatin-like phospholipase Biochemistry His6 hexahistidine tag Mice SMT3 small ubiquitin-like modifier Transacylation mATGL mouse ATGL Protein purification Sf9 Cells MAG monoacylglycerol CV column volume G0S2 NusA transcription termination/antitermination protein biology Chemistry Hydrolysis ABHD5 Ni2+ nickel (II) ion Recombinant Proteins TRX thioredoxin MTBE methyl tert-butyl ether CGI-58 comparative gene identification-58 DGAT diacylglycerol acyltransferase NTA nitrilotriacetic acid HSL hormone-sensitive lipase Research Article SEC size-exclusion chromatography LD lipid droplet TAG triacylglycerol MBP maltose-binding protein Spodoptera TEV tobacco etch virus GST glutathione-S-transferase Animals Humans Lipase Molecular Biology PNPLA2 Diacylglycerol kinase ATGL adipose triglyceride lipase CGI-58 OPPF Oxford Protein Production Facility FA fatty acid TCEP Tris(2-carboxyethyl)phosphine Cell Biology HEK293T human embryonic kidney 293T Fusion protein Monoacylglycerol lipase ONC overnight culture HEK293 Cells Adipose triglyceride lipase lipolysis biology.protein BSA bovine serum albumin G0S2 G0/G1 switch gene 2 DAG diacylglycerol |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/j.jbc.2021.101206 |
| Popis: | 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 human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal 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 copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 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 triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity 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. |
| Databáze: | OpenAIRE |
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