Figure 7

Detergent and urea solubilization and purification of recombinant DGTA1 from insoluble fraction by Ni-NTA affinity chromatography. The insoluble fraction (10,000g pellet) of E. coli was solubilized with 7 detergents (0.5% final concentration, 4°C, 1 h) followed by centrifugation at 20,000g for 10 min. The 20,000g supernatant was used for affinity purification with Ni-NTA beads. Recombinant DGAT1 was detected by immunoblotting using anti-MBP-hTTP antiserum. (A, C) solubilized fractions, (B, D) purified fractions. Lane 1, protein size standards; Lane 2, Brij 35; Lane 3, CHAPS; Lane 4, NP-40; Lane 5, SDS; Lane 6, Triton X-100; Lane 7, Tween 20; Lane 8, Tween 80; Lanes 9 to 13, 0, 0.1, 0.3, 0.5 and 1% SDS, respectively. (C, D) Lane 1, protein size standards, Lanes 2-8, 0, 0.1, 0.3, 0.5, 1, 1.5 and 2% Triton X-100, respectively; Lanes 9-12, 0, 2, 4 and 6 M urea, respectively. Each lane was loaded with equal amounts of proteins corresponding to those before solubilization. The full-length rDGAT1 is marked with an arrow.