Ponente
Descripción
The main function of protease inhibitors is to prevent undesirable proteolysis. CmPI-II is a 50 amino-acids-long serine protease inhibitor isolated from the marine snail Cenchritis muricatus. It is a tight binding inhibitor of trypsin (Ki = 1.1 nmol/ L), human neutrophil elastase (Ki = 2.6 nmol/ L), subtilisin A (Ki = 30.8 nmol/L) and porcine pancreatic elastase (Ki = 145 nmol/L). It belongs to the Kazal family and due to the novel topology of one of its disulfide bridges, it defined the group 3 within the non-classical inhibitors. This molecule represents an exception in terms of specificity due to its ability to strongly inhibit elastases and trypsin. Therefore, the study of its structure-function relationship is of interest. The construction of combinatorial libraries of protease inhibitor mutants, displayed on filamentous phages, is a very useful technique for studying structure-function relationships. The process begins with functional phage display of the wild-type inhibitor. In the present work, CmPI-II was phage displayed fused to the phage coat protein PIII. In ELISA experiments, the ability of CmPI-II, displayed on phage, to bind trypsin and pancreatic elastase was demonstrated. Future work is directed to evaluate the reactivity with HNE and subtilisin A, and to design and construct a combinatorial library containing CmPI-II mutants, to isolate those able to selectively bind the target proteases. These results will provide information on the relative importance of CmPI-II residues for binding to each target enzyme.
