Fig. 5. (A) Samples of soluble crude extracts prepared from E.coli cells transformed with plasmids pET21a (1), pET21a-His₆-SSU72 (2), pET21a-SSU72-C15A (3) or pET21a-SSU72-3319 (4) were run on SDS–PAGE gels. The position of protein Ssu72 is indicated by an asterisk. (B) Phosphatase activities present in samples (1 mg) of soluble crude extracts prepared from E.coli cells transformed with plasmid pET21a carrying either no insert (diamond), wild-type SSU72 gene (square), mutant ssu72-C15A (triangle) or mutant ssu72-3319 (cross). Samples were assayed for their ability to cleave pNPP (20 mM) after 20 min of incubation at 28°C. (C) Purification of His₆-tagged Ssu72 protein. A sample of the purified protein was run on an SDS–PAGE gel. (D) Phosphatase activity of the purified His₆-Ssu72 protein. Samples of purified Ssu72 (45 µg) were incubated as described in Figure 4A (diamond), in presence of 100 µg/ml BSA (square) or at 37°C (triangle). (E) Ortho-vanadate inhibition. Samples (45 µg) of purified His₆-tagged Ssu72 protein were incubated with 20 mM pNPP during 20 min at 28°C, with an increasing concentration of ortho-vanadate.