Figure 7.

Analysis of the cleavage specificity of human gzm K by the use of recombinant protein substrates. The cleavage of substrates by human gzm K are shown in (A-D). The sequences of the target regions and cleavage time (minutes) were indicated above the lanes of different samples. The bands between 26kDa and 34kDa represent un-cleaved substrates and those between 10kDa and 17kDa represent cleaved substrates. The difference in size of cleaved bands was caused by the His₆-tag of one of the Trx molecules. All protein gels were analyzed using the UN-SCAN-IT Gel Analysis Software from Silk Scientific Inc. (Orem, Utah USA). By the densitometric scanning, shown in (E), and comparing number of minutes to get to the same percentage of cleavage we estimate a major approximately 30 times lower cleavage rate for variant 1 (V1), a six times difference for V2, no difference for V3, a 10-15 fold difference for V4, a three-fold difference for V5, a 3-4 fold for V6 and a 50 fold difference for V7. The high reproducibility in the assay can also be seen from the four samples of the consensus sequence that was run at different times, C1, C2, C3 and C4, which all gave almost identical scanning results, all shown in blue.