Expression and characterization of metallopeptidases neprilysin (EC 3.4.24.11) and endothelin converting enzyme 1 (EC 3.4.24.71) in Pichia pastoris, followed by the identification of new substrates and/or inhibitors

• Grant number: 06/04409-7
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2007
• End date: January 31, 2010
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Vanessa Rioli
• Grantee: Vanessa Rioli
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

The present project intends to develop an efficient system for expression and purification of two important therapeutics targets, metallopeptidases ECE-1 (Endotelin 1 converting enzyme; EC 3.4.24.71) and NEP (Neprilysin; EC 3.4.24.11). The expression of these recombinant enzymes will be carried through in Pichia pastoris. Also, it will be our objective to inactivate these enzymes by site-directed mutagenesis. The inactive proteases will be used in the identification of possible new natural substrate and/or natural inhibitors for these enzymes, as previously described (Rioli et al., 2003; patent pending from Rioli, V and Ferro, E.S. INPI PI0301511-4, from 16/05/2003) ), from complex mixtures of poison venoms and animal tissues. for the proponent. The use of this strategy for the isolation of new substrates/inhibitors is supported also by recente evidences that the largest number of drugs recently approved, or waiting approval, for the FDA are peptides of natural origin (Watt, 2006). Moreover, it is important to mention that this strategy (to use inactive enzymes to isolate new substrates or competitive inhibitors) was originally developed during my PhD and could be more efficient than combinatorial libraries searching for new therapeutic targets. (AU)