Figure 4.

Charge substitutions of surface residues did not restore the inhibitory effect of H69E on protease autoprocessing. (A) Schematic presentation of the mature protease dimer (PDB 2PK6) with the surface residues that were tested in this report highlighted in red or green and histindine 69 in blue. (B) The pGEX-3X derived plasmids encoding for GST-TFR-PR-FLAG fusions bearing the indicated mutations were introduced into E. coli BL21(DE3) and induced for protein expression. The total lysates were prepared as described (Materials and Methods) and subjected to western blot analysis. A mouse anti-FLAG antibody was used to detect the full length precursor fusion, intermediates and mature protease (PR-FLAG). The denoted protein markers are in kDa for reference.