Expression in Escherichia coli, purification, refolding and structural analysis of the coat protein of rupestris stem pitting associated virus

• Grant number: 11/23680-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: April 02, 2012
• End date: July 01, 2012
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Raghuvir Krishnaswamy Arni
• Grantee: Ricardo Barros Mariutti
• Supervisor: Christian Betzel
• Host Institution: Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP). Campus de São José do Rio Preto. São José do Rio Preto , SP, Brazil
• Institution abroad: Universität Hamburg (UHH), Germany
• Associated to the scholarship: 10/19259-6 - Expression in Escherichia coli, purification, refolding and structural analysis of proteins of the movement and coat protein of Rupestris stem pitting associated virus, BP.DR

Abstract

The grape is the most economically important fruit in the world. The berries of the grape are the basis for the high value of wines and other alcoholic beverages. Rupestris stem pitting (RSP), a component of the rugose wood complex, is one of the most widespread graft transmissible grapevines (Vitis spp.) virus. The RSP is characterized by the presence of small pits in the woody cylinder below the point of inoculation by chip budding on Vitis rupestris. A virus named Ruspestris stem pitting-associated virus (RSPaV), has been associated with the disease . The virion has a positive sense, single stranded, polyadenylated RNA genome of 8.7kb in size and a coat protein of 28kDa.This disease is transmitted by grafting and is one of the most widespread viruses of grapevine in the world, being described in Brazil in the late '60s. Due to the difficulty of purification of viral particles from infected grapevines, large-scale expression of viral proteins in bacteria is a promising strategy for understanding their properties. This work aims to produce in the native state, protein coat protein (CP) of 28 kDa, encoded by RNA rupestris stem pitting associated virus. From the expression in Escherichia coli, this work aims large quantities (milligrams) of proteins with high degree of purity for crystallization trials and the determination of their three-dimensional structures at high resolution. Studies of crystallization and X-ray analysis could provide data on the structure of viral particles that are scarce.Some results as an expression and purification of the protein in native conditions have been obtained. The circular dichroism showed that the protein is structured and several crystallization trials were made. (AU)