Localization of recombinant DGAT1 in the insoluble fraction and membranes of E. coli. (A) Trace amount of rDGAT1 was in the soluble fraction. The 10,000g supernatant of E. coli transformed with pMBP-DGAT1-His was loaded onto an MBPTrap HP column. The column was washed extensively with amylose resin wash buffer. The bound proteins were eluted with amylose resin elution buffer containing 20 mM maltose. Proteins were separated by 10% SDS-PAGE. Recombinant DGAT1 was identified by anti-MBP-hTTP antibodies. (B) The majority of rDGAT1 was in the insoluble fraction. The 10,000g pellet of E. coli transformed with pMBP-DGAT1-His was subject to extensive sonication followed by centrifugation at 10,000g. This supernatant was loaded onto an MBPTrap HP column. Separation and detection of the recombinant protein was identical to those in panel A. (C) Localization of rDGAT1 in the membranes. The 10,000g supernatant was centrifuged at 100,000g. Proteins in the 100,000g supernatant (cytosol) and the pellet (membranes) were separated by 4-20% SDS-PAGE, transferred onto a nitrocellulose membrane, and detected by immunoblotting with anti-MBP-hTTP antibodies. S100, 100,000g supernatant (cytosol); P100, 100,000g pellet (membrane). The full-length rDGAT1 is marked with an arrow. (D) Purification of MBP from the 10,000g supernatant of E. coli transformed with pMAL-c2X as a positive control for amylose resin affinity chromatography.