Transformation of the Mpr1 prodomain mutations into <i>mpr1Δ</i> strain shows the expression of Mpr1 at the protein level.

(A) The plasmid vector containing Mpr1 with the point mutations (D353A and D355A) at the predicted cleavage sites was cloned into the mpr1Δ deletion strain. The endogenous promoter and terminator of Mpr1 was used to regulate the expression of Mpr1. A Myc-tag was added to the C-terminus of Mpr1 for protein detection. The wild type HExxH and ExxxD conserved motifs were kept intact in order to maintain autocatalytic activity. (B) Genomic DNA isolation and PCR confirmed the transformation of the Mpr1 prodomain mutations (MPR1ProMut-Myc) into the mpr1Δ strain. Bands at ~2.2 Kb, corresponding to the size of Mpr1 plus the Myc-tag, were detected in the reconstituted strain (MPR1WT-Myc) and the mutant strain (MPR1ProMut-Myc)) but not in mpr1Δ strain. The wild type H99 strain and a plasmid containing Mpr1 cDNA were used as controls for PCR. The genomic DNA of H99 encoding Mpr1 displayed a band size of ~2.7 kb as expected. (C) Western blot analysis using an anti-Myc-tag antibody revealed expression of Mpr1 in MPR1WT-Myc and MPR1ProMut-Myc. Distinct bands at ~85 kDa and ~40 kDa, which correspond to the size of immature and mature forms of Mpr1 respectively, were observed in the reconstituted strain (MPR1WT-Myc). In contrast, the mature form of Mpr1 was not detected in the MPR1ProMu-tMyc strain suggesting that the prodomain mutation prevented processing of a fully mature Mpr1. GAPDH was used as an loading control.