FIGURE 4.

Overproduction, purification and PPIase activity of Cj0694. (A) Lane 1; Cj0694 lacking the N-terminal membrane anchor and with a C-terminal his-tag initially purified by Ni-NTA affinity chromatography (monomer molecular weight 54.8 kDa). Lane M; molecular weight markers. (B) Further purification of Cj694 by DEAE anion-exchange chromatography with elution from 0 to 1 M NaCl. Lanes 1–7 are samples taken across the UV-absorbing peak eluted from the column and show a single major band on SDS-PAGE after staining with Coomassie Blue. Note that the migration of the protein is affected by the salt present in the elution buffer and the apparent molecular weight is higher than in (A). Lane M; molecular weight markers. (C) PPIase activity of Cj0694 demonstrated by refolding of RCM-RNaseT₁ in the presence of 4 M NaCl (see Materials and Methods) either without or with the addition of purified Cj0694 as shown. The purified periplasmic chaperone PEB4 was used as a positive control. The fluorimeter was set to zero at the time of dilution, so that the increase in fluorescence results from the uncatalyzed (blue progress curve) or chaperone catalyzed (green, orange, and red progress curves) refolding process. Results shown are a single representative experiment.