Expression and purification of recombinant cruzain from E. coli DH5a

• Grant number: 13/21102-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2014
• End date: June 30, 2014
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Carlota de Oliveira Rangel Yagui
• Grantee: Gustavo Muneo Takata
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Chagas Disease is an endemic disease caused by the parasite Trypanosoma cruzi with severe consequences for the affected individuals. Cruzipain is a cystein-protease expressed by T. cruzi that was demonstrated to be related with pathogenicity and virulence and, therefore, considered a potential therapeutic target. This enzyme was already expressed in E. coli strain DH5a, as the recombinant form cruzain. The aim of this project is to optimize the cruzain production and, therefore, to have enough amounts to be used in biological assays of potentially anti-T. cruzi compounds (cruzain inhibitors). Initially, the effect of the temperature and time on the enzyme induction of expression by E. coli will be investigated. Following, the best conditions for cruzain purification by Fast Protein Liquid Chromatography (FPLC) will be defined. The kinetic parameters of the enzyme produced ((Km and Vmax) will also be determined and compared to values available in the literature.