Abstract
BackgroundDiacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in E. coli had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in E. coli.ResultsAn expression plasmid containing the open reading frame for tung tree (Vernicia fordii) DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in E. coli BL21(DE3). Immunoblotting showed that the recombinant DGAT1 (rDGAT1) was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification.ConclusionsThis study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.
Highlights
Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms
Degradation of recombinant DGAT1 in the soluble fraction of E. coli The expression of recombinant protein MBP-DGAT1-histidine residues (His) (rDGAT1) was induced by IPTG in E. coli strain BL21(DE3) and detected by immunoblotting using anti-MBP-hTTP and anti-MBP-mTTP polyclonal antibodies, which were raised in rabbits against purified recombinant human and mouse TTP proteins fused to MBP [2,3]
Detergent and urea solubilization and purification of recombinant DGAT1 from insoluble fraction As the results indicated that rDGAT1 was associated with the insoluble pellet, an attempt was made to solubilize the recombinant protein from the 10,000g pellet with seven different detergents (Brij 35, CHAPS, NP-40, SDS, Triton X-100, Tween 20 and Tween 80) and urea followed by purification with Ni-NTA affinity chromatography
Summary
Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in E. coli had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. Diacylglycerol acyltransferases (DGATs) are responsible for the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT isoforms have nonredundant functions in TAG biosynthesis in plants such as tung tree (Vernicia fordii) [10] and animals such as mice [13]. The fact that DGAT1 is unable to compensate for the deficiency in DGAT2 knockout mice indicates that each DGAT isoform has unique functions in TAG biosynthesis during mammal development
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