Proteases are enzymes involved in several biological processes such as vasoactive peptides release, blood coagulation, fibrinolysis and complement cascade, and are related to serious pathological processes. Elastase is one of the most destructive enzymes in the body, and without control by endogenous inhibitors, this enzyme may cause damages, as pulmonary edema. The search for new elastase inhibitors is justified, because the known inhibitors are harmful due to secondary effects. Protease Inhibitors are essential to life maintenance and, in plants, the leguminous have an enormous quantity of these proteins in seeds, fruits and roots. Our group has been working in the isolation and characterization of different enzymes and inhibitors from Caesalpinia echinata (brazil wood) seeds. Recently, we purified from these seeds an one chain 23 kDa Kunitz-type elastase inhibitor. This protein (CeEI) presented high inhibitory activity to human neutrophil elastase (Ki = 1.9 nM).
The aims of the present study were: (i) the cDNAs cloning of two elastase inhibitors from C. echinata; (ii) The expression and purification of the recombinant inhibitors.
The CeEI gene was cloned by RT-PCR using RNAm prepared by C. echinata (CeEI) premature seeds. PCR amplified DNA fragment (700 bp) using a degenerated oligonucleotide based on the CeEI N-terminal amino acid sequence was ligated into the pGEM-T easy vector and sequenced. The translated amino acid sequence of two clones presented two putative Kunitz type serine protease inhibitors. The genes were sub-cloned into pET-14b vector (the protein is fused to a His-Tag) and the recombinant proteins expressed by E. coli Rosetta Gami. The recombinant proteins were purified by affinity (Ni-NTA agarose column) and ion-exchange (Hi-Trap Q) chromatographies. The purified proteins presented dissociation constants for human neutrophil elastase (K_i = 0.7 nM) and chymotrypsin (K_i of 145 and 31.2 nM). The comparison of the CeEI amino acid sequences with other proteins allowed us to classify them into the Kunitz-type family of serine protease inhibitors.