Figure 8.

Analysis of C-terminal variants CGEP-C1 and CGEP-C2 using rGST-CGEP fusion proteins. A, C-terminal sequence of CGEP and the CGEP-1 and CGEP-C2 variants. W907 is the conserved residue across CGEP homologs in photosynthetic organisms, E929A and D931S are the mutations in CGEP-C1 and E946A, and E949A and E951A are the mutations in CGEP-C2. The arrow indicates the cleavage site of the C-terminal prosequence in wild type (in vitro and in vivo) as shown in Figure 5. Peptides matched for the C-terminal regions are provided in Supplemental Table S2. B, Comparison of protein size of rGST fusion variants of CGEP (wild type, S781R, C1, and C2) shows that rCGEP-S781R and rCGEP-C2 are a few kilodaltons larger than rCGEP and rCGEP-C1, indicating their lack of autocatalytic C-terminal processing. Top shows the Coomassie-stained gel; middle shows the Ponceau-stained blot; and the bottom shows the immunoblot with anti-CGEP serum. C, Degradation assay of insulin and casein using rGST-CGEP-C2 showed that GST-CGEP-C2 can completely degrade the 10-kD insulin, but it cannot degrade the larger 25-kD β-casein. Incubation times (h) are shown.