Figure 5

Purification of recombinant DGAT1 from E. coli with Ni-NTA and amylose resin affinity chromatography. (A, B) Ni-NTA affinity purification. The 10,000g supernatant was mixed with Ni-NTA affinity beads. The beads were washed five times followed by eluting the beads with imidazole solution containing 50 mM (Elution 1), 100 mM (Elution 2), 150 mM (Elution 3), 200 mM (Elution 4), 250 mM for 5 times (Elutions 5.1-5.5), and 1000 mM (Elution 6). Proteins were separated by 4-20% SDS-PAGE, stained with Coomassie brilliant blue (A) or transferred onto nitrocellulose membranes for immunoblotting with anti-MBP-hTTP antibodies (B). (C, D) Amylose resin affinity purification using Ni-NTA affinity-purified fractions. The fractions purified by Ni-NTA affinity beads as shown in panes A and B were pooled, centrifuged, and loaded onto an MBPTrap HP column. SDS-PAGE separation and immunoblotting detection of the recombinant protein was identical to those in panels A and B. S, 10,000g supernatant; P, 10,000g pellet. The full-length rDGAT1 size is marked with an arrow.