Clonning, expression and structural studies of the GTPase domain from recombinat sseptins from Schistosoma mansoni

• Grant number: 12/12113-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2012
• End date: November 30, 2014
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Ricardo de Marco
• Grantee: Yuri Volpato Santos
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

This study aims to express and characterize the GTPase domain of septins from the trematode flatworm Schistosoma mansoni. This parasite is the etiologic agent of Schistosomiasis, a disease that affects about 210 million people in 74 countries. The septins are a conserved family of GTP-binding proteins, which were first associated with the process of cytokinesis in yeast budding. Today, it's known this protein plays an important role in a variety of cellular functions, like cell morphology, neuronal polarity, and vesicle trafficking. We have cloned the GTPasic domain of septins 7.1 and 7.2 from Schistosoma mansoni, which were named Smspt7.1G and Smspt7.2G. Polypeptides representing both domains will be expressed in E. coli rosetta (DE3)using the expression vector pET28a(+). The expression will be performed at 18°C and we hope to obtain both recombinant domains in a soluble form. The process of purification will comprise affinity chromatography on Ni-NTA resin followed by size exclusion chromatography on a Superdex 200 column. The purified domains will be submitted to Circular Dichroism (CD) Spectroscopy. Once we observe a folded secondary structure for both of them we will realize activity assays performed with GTP as subtract to evaluate the hydrolytic capacity of these domains. Preliminary crystallization assays will be performed and if the formation of crystals occurs they will be submitted to X-ray diffraction.(AU)