Figure 5. Purification of recombinant DGAT2 from E. coli with tandem Ni-NTA and amylose affinity beads.

(A) Ni-NTA purification. The 10,000g supernatant was mixed with Ni-NTA affinity beads. The beads were washed five times. Proteins bound to the beads were eluted by imidazole solution containing 50, 100, 150, 200, 250, 1000 mM (Elutions 1–6). Proteins were separated by 4–20% SDSPAGE and stained with Coomassie brilliant blue (A, top) or transferred onto nitrocellulose membranes for immunoblotting with anti-MBP-hTTP antibodies (A, bottom). The full-length rDGAT2, MBP, and the potential dimer of the full-length rDGAT2 are marked with arrows. H, homogenate, S2, 2,000g supernatant, S10, 10,000g supernatant, U, unbound fraction, B, Ni-NTA beads after 6 imidazole elutions. (B) The fractions purified by Ni-NTA affinity beads as shown in A were mixed with amylose resin affinity beads. The beads were washed five times. Proteins bound to the beads were eluted by amylase resin elution buffer containing 20 mM maltose (lanes 1–5) and three times with 0.5 M NaOH (lanes 6–8). Proteins were separated by 4–20% SDS-PAGE and stained with silver reagent (B, top) or transferred onto a nitrocellulose membrane for immunoblotting with anti-MBP-hTTP antibodies (B, bottom). The full length rDGAT2 and the potential dimer of the full-length rDGAT2 are marked with arrows. E4, proteins eluted with 200 mM imidazole solution from Ni-NTA beads (Figure 5).