Figure 1.

Design, expression and characterization of mutated PR3. (a) Design of mutated PR3. Schematic representation of the PR3 variants. The signal sequence, prosequences, and C-terminal sequence are shown as grey, black and speckled boxes, respectively. Exons are indicated with the exon number in the box. The amino acids H⁴⁴, D⁹¹ and S¹⁷⁶ forming the catalytic triad and the two glycosylation sites on N¹⁰² and N¹⁴⁷ are marked at the wild-type PR3. PR3 variants are listed below, the introduced point mutations are written in parenthesis. (b) Enzymatic activity of rPR3; 500 ng PR3 variants were incubated with 500 ng PR3H44Q-Ang fusion protein as substrate for 1, 2, 4 and 20 hr at room temperature. Then, protein decomposition was measured by SDS–PAGE analysis. Proteins were visualized by Coomassie staining. (c) Affinity of rPR3 variants to anti-PR3 mAb. The rPR3 constructs were tested by direct ELISA using WGH1, WGM2, or WGM3 mAb. Bound antibody was detected by horseradish peroxidase-conjugated goat anti-mouse antibody. Therefore rPR3 was coated with a final concentration of 100 ng. The anti-CD30 scFv fragment was used as a non-specific control. Error bars represent SD values of triplicates.