Advanced search
Start date
Betweenand

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

Grant number: 12/12113-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): October 01, 2012
Effective date (End): November 30, 2014
Field of knowledge:Biological Sciences - Biophysics
Principal Investigator:Ricardo de Marco
Grantee:Yuri Volpato Santos
Home 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 from Schistosomiasis, a disease that affects about 210 million people in the 74 countries. The septins are a conserved family of GTP-binding proteins, which were first associated to the process of cytokinesis in yeast budding. Today, it's known this proteins play 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 an 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 this domains. Preliminary crystallization assays will be performed and if the formation of crystals occur they will be submitted to X-ray diffraction.