Recombinant SmKI-1 Kunitz Domain mutants present enhanced neutrophilic elastase inhibitory activity in vitro when compared to KD.

Soluble recombinant mutants RL-KD and EA-KD were tested towards their neutrophilic elastase (A), trypsin (B) and plasmin (C) inhibitory activities when compared to KD. Enzymes (100 nM) were incubated at 37°C with soluble recombinant RL-KD, EA-KD and KD proteins (100nM or 300 nM) in the presence of each enzyme’s substrate. Bovine serum albumin (BSA) was used as negative control. Elastase, trypsin and plasmin inhibition activities were detected over one hour, four hours and twenty minutes of incubation with the soluble proteins, respectively. Bars indicate the mean activity for each enzyme ± standard deviation. * represents statistical difference (p value < 0.05) when compared to control group. # represents statistical difference (p value < 0.05) when compared to rKD group. rRL-KD mutant presented the best performance towards inhibition of neutrophil elastase in vitro, while rEA-KD presented enhanced inhibitory activity towards plasmin. These results are representative of three independent experiments.