Figure 3.

Purification and the Activities of the Wild-Type DHPS (DHPS^WT), p.Asn173Ser (DHPS^N173S), and p.Try305_Ile306del (DHPS^Y305_I306del) Variants

The recombinant enzymes were expressed in BL21(DE3) cells and the enzyme activity was measured as described under Material and Methods.

(A) Coomassie Blue-stained patterns of proteins in total lysates (lanes 1–4), lysate supernatants (lanes 5–7), or purified enzymes (lanes 8–10) are shown. Lysate proteins of BL21(DE3) not transformed with a DHPS expression vector is shown in lane 1, for comparison with those of DHPS-overexpressing lysates.

(B) Comparison of enzyme activities. The enzyme activity assays were performed with total lysates (0.2 μg proteins in each sample), lysate supernatant (0.1 μg proteins in each sample), or purified DHPS enzymes (0.02 μg each sample) as described under Material and Methods. The activities of the mutant enzymes are expressed as percent of the wild-type enzyme in each group. The experiments were repeated three times with similar results. Data from a representative experiment with duplicate samples is shown with error bars.