Fig. 2. Aggregation and formation of a proteinase-K-resistant core in recombinant HET-s(157-289). (A) Recombinant HET-s(157-289) aggregates in vitro and catalyses the aggregation of full-length HET-s. (top) Time course of aggregation at 0.2 mg ml^–1 at 20°C. (bottom) Aggregation of recombinant full-length HET-s protein at 3 mg ml^–1 inoculated with an aliquot (0.1 mg ml^–1) of aggregated HET-s(157-289) (triangles) or buffer alone (squares). (B) Circular dichroism spectra of 20 µM recombinant HET-s(157-289) at pH 8 in the soluble (black triangles) and aggregated (white squares) states. (C) Time course of limited proteinase-K digestion of soluble (top) and aggregated (bottom) recombinant HET-s(157-289) protein analysed by SDS-PAGE followed by Coomassie-Blue staining. Digestion times are given in minutes. The last lane (218-289) corresponds to the recombinant HET-s(218-289) peptide. Size of molecular weight markers (M lane) is given in kDa. (D) Mass spectrum of the proteinase-K-resistant material. The y axis gives intensity in arbitrary units, the x axis gives mass-to-charge (m/z) ratios in Daltons per unit charge. The peak at 8650.81 Da (*) corresponds to an internal control (recombinant 218-289 peptide with a six-histidine extension). The major peak at 8520.07 was identified as the 218-289 fragment (with six histidines). The 130.74 Da difference in the measured average mass between the control recombinant 218-289 peptide (*) and the major peak is caused by the recombinant peptide displaying an additional N-terminal methionine residue (theoretical mass difference 131.20 Da).