Expression, purification, refolding and crystallization of the coat protein from Rupestris Stem Pitting associated Virus

• Grant number: 08/00309-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2008
• End date: December 31, 2008
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Mário Tyago Murakami
• Grantee: Joice Helena Paiva
• 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

Abstract

The cultivation of the grapevine is exposed to a large number of diseases which can generate significant losses in agriculture worldwide. Viruses are the most responsible for these diseases. One example, is characterized by grapevine rugose wood, it is a complex of four diseases, including the highlight of this work that is the rupestris stem pitting (RSP) promoted by Rupestris stem pitting associated virus (RSPaV). The genome of this virus is composed of five open reading frames, and the fifth ORF is related to the viral coat protein which has a molecular mass of approximately 28 kDa. The infected plant displays a declined in the growth, quality and production of fruits. The first symptoms is evident after periods up to two years which difficult the detection and diagnostic of the rupestris stem pitting. Moreover, there is an irregularity in the distribution of the virus in the plant that also varies with the season. Thus, there is a need for a more efficient method in the detection of the virus. Structural studies of the coat protein can significantly shed light in the molecular mechanisms involved in the recognition and virus transmission providing tools and knowledge for the development of a more effective method in the detection of the virus in the plant. So far, none three-dimensional protein structure related to replication, cell-to-cell movement, recognition and infection from RSPaV has been solved. Thus, the aim of this project is the scale up production of recombinant coat protein from RSPaV by applying molecular biology and biochemical techniques to further structural investigations by X-ray crystallography.