HF3, a P-III class metalloproteinase isolated from the B. jararaca venom, is a potent hemorrhagic toxin, which degrades extracellular matrix proteins and inhibits platelet aggregation. A 2045 bp cDNA encoding HF3 indicated that it is a multidomain protein composed of a pro-domain, a catalytic domain with the characteristic zinc binding sequence, followed by disintegrin-like and cysteine-rich domains. It is known that metalloproteinases play a relevant role in the pathogenesis of viperid venom-induced local tissue damage including inflammatory reactions. We have shown that native HF3 and its recombinant disintegrin-like/cysteine-rich domains (DC/HF3) were able to increase aMb2-mediated phagocytosis of opsonized-zymosan particles by macrophages. Recently, our studies on the role of the cysteine-rich domain of atrolysin A, a P-III class metalloproteinase from Crotalus atrox venom, pointed to its function as a cell-surface-receptor-binding site and/or a substrate recognition exosite. In order to define further the role of the cysteine-rich domain of HF3 as an exosite of this multidomain metalloproteinase involved in its interaction with specific targets, in this work the cysteine-rich domain of HF3 (C/HF3) was expressed as a recombinant protein. The cDNA sequence coding for the cysteine-rich domain (an amino acid sequence of 113 residues including 12 cysteines) was subcloned in the expression vector pGEX-4T2, which allows the expression of soluble recombinant proteins in fusion with Glutathione S-transferase. E. coli DH5a cells were transformed with the resultant expression plasmid pGEX-4T2-C/HF3 and protein expression was induced with 0.5 mM IPTG for 4h. The recombinant fusion protein GST-C/HF3 purified by affinity chromatography on Glutathione Sepharose 4B showed to be essentially homogeneous by SDS–PAGE and by Western blot using an anti-HF3 antibody. The authenticity of the recombinant-protein was also analyzed by mass spectrometry.
Financial Support: FAPESP