Fig. 8.
Analytical size-exclusion chromatogram of ST3:NSP495–146 under different conditions. (a) Lysis of cells in NaAc buffer (pH 5.6) (condition A). (b) Lysis in the absence of calcium (condition B). (c) Lysis in the presence of 10 mM calcium (condition C). (d) Lysis and purification in the presence of 0.1 mM calcium (condition D). (e) Lysis and purification in the presence of 0.1 mM magnesium (condition F). (f) SEC of the peptide purified under condition B at concentrations of 20 mg/ml and 5 mg/ml and under condition D at a concentration of 5 mg/ml. The NSP4 peptide in the bacterial cell lysate was precipitated in 40% ammonium sulfate. All subsequent purification steps, irrespective of cell lysis conditions, were carried out under physiological buffer conditions in the absence or presence of added calcium or magnesium. The protein was purified by ion-exchange chromatography followed by size-exclusion chromatography, and the molecular masses were determined as described in Materials and Methods. Vo, void volume.