Figure 5.

Analysis of the selectivity of human gzms A and K against substrates with an Arg or a Lys in the P1 position. In (A), the overall structure of the recombinant protein substrates used for the analysis of the cleavage by gzm A is depicted. The sequences analyzed were positioned between two thioredoxin molecules with a His₆-tag attached to the C terminal of the second Trx molecule. Two unique restriction sites (BamHI and SalI) were used for the insertion of the target sequences between the two thioredoxin molecules. The major steps of the analysis are shown in (A). A schematic example of this type of analysis is depicted in (B) explaining various possibilities of the cleavage patterns. (C) shows the cleavage with Arg or Lys containing substrates with gzm A and gzm K The first two substrates with gzm A and the following two substrates with gzm K The substrates used for cleavage are the consensus substrates for the respective enzyme with an Arg in the P1 position and the second substrate are a substrate where the Arg has been exchanged for a Lys, marked in red. All protein gels were analyzed using the UN-SCAN-IT Gel Analysis Software from Silk Scientific Inc. (Orem, Utah USA). By scanning we measure the remaining amount of the 2xTrx protein in percent compared to the starting material after 15, 45 and 150 minutes of cleavage. The scanning result is shown in (D). By the densitometric scanning and comparing number of minutes to get to the same percentage of remaining material we estimate a 15 times difference in cleavage efficiency between an Arg and a Lys in the P1 position for gzm A and a 3.75 times difference for gzm K.