Figure 5
Autoprocessing of precursors with different C-terminal tags. All fusion precursors were derived from GST-M2-PRNL-Flag for specific examination of P site cleavage reaction. (A) The sequences at the junction between the PR and the C-terminal tag of some constructs are listed. (B) Autoprocessing of precursors carrying different small peptide epitopes or no tag were analyzed using a mouse epitope-specific antibody (Flag, Myc, HA), polyclonal rabbit anti-V5, or anti-PR as underlined at left, and GST primary antibodies for dual visualization. Because the signal detected by anti-PR antibody was weak, the boxed region was enhanced to show the mature protease (bottom). Autoprocessing of precursors fused to a GCN4 motif (C) or to reverse transcriptase (RT) in which the native cleavage site between PR and RT remained cleavable (E) or was mutated to uncleavable (D) were also compared. Asterisks indicate self degradation IC50 of the PR-containing product; open triangles indicate the apparent IC50 of the cleavage reaction. The expected products from P site cleavage reaction are connected by a solid line.