Molecular cloning and expression of Trypanosoma brucei U5-200K protein for tandem purification experiments

• Grant number: 13/26996-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2014
• End date: November 30, 2015
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Otavio Henrique Thiemann
• Grantee: Camila Maria dos Santos Boralli
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 08/57910-0 - National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases, AP.TEM

Abstract

Given the great medical importance of the Trypanosomatidae family, since parasites of this family are capable of causing lethal disease in their hosts, have been investigated several molecular processes such as the processing of pre-mRNA by Splice-Leader (SL trans-splicing) required to resolve polycistronic RNA transcripts into mature mRNAs. This mechanism is absent in vertebrates, in which the cis-splicing process occurs, therefore the trans-splicing proteins are considered important target parasite-specific proteins. The spliceosome is a protein complex that interacts with nuclear SLRNA and pre-mRNA to catalyze trans-splicing, comprising small nuclear ribonucleoprotein particles (U1 , U2, U4 , U6 and U5 snRNPs) formed by protein factors and snRNAs specific members. The spliceosome requires rearrangements to obtain the catalytic conformation with the pre-mRNA. The protein U5-200K, U5 snRNP particle component T. brucei has two domains: a helicase DEAD/Deah box and a Sec63 domain. The C-terminal region of this protein does not retain other Sec63 domain, as in yeast and mammals, and its function has not yet been identified. In yeast, the Sec63 N-terminal domain cassette Brr2 is an integral part of the active site, allowing the disruption of the U4/U6 duplex during catalytic activation of the spliceosome. This project aims to amplify the ORF of U5-200k from T. brucei genomic DNA. Molecular cloning vector pC-PTP-neo which allows expression of the protein of interest fused to the tandem epitope tag (PTP, P, two protein domains A, T, a site TEV protease, P, and domain Protein C). We intend to obtain different constructs, starting with the native U5 - 200K and mutant in the latter case, the aim is to obtain proteins that do not have each specific domain. After establishing the T. brucei strains expressing U5-200K and mutant PTP-products, the main object of this project aims to study the influence of U5-200K and its domains in the formation of the U5 snRNP complex or possibly other participants in the splicing reaction (AU)