Establishment of PTP-tag method for the purification of protein complexes containing the proteins FLB-3 and RUV-1 of Neurospora crassa

• Grant number: 16/14384-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2016
• End date: July 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Daniela Luz Ambrósio Breisch
• Grantee: Allan Pradelli Roldão
• Host Institution: Instituto de Química (IQ). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated research grant: 13/24705-3 - The filamentous fungus Neurospora crassa as a model organism for the functional characterization of proteins/transcription factors that regulate the carbohydrate metabolism, AP.TEM

Abstract

The FlbC transcription factor was described, originally, in A. nidulans as an asexual development regulatory protein, triggering the conidiation process through the activation of a cascade of genes and, until now, it has not been very well functionally characterized in other filamentous fungi. Previous results obtained with fungi N. crassa showed that the protein ortholog FlbC, called FLB-3, regulates sexual and asexual development of the fungus and also participates in the regulation of the reserve carbohydrates metabolism, glycogen and trehalose. Considering the previous results obtained with this transcription factor in our laboratory, the investigation of FLB-3 interaction with other proteins will be important for the functional characterization. The TAP-tag (Tandem Affinity Purification) method has been used in variety organisms for the purification of protein complexes present in extracts through the use of a tag fused to a target protein. This process occurs in two purification steps, which results in the removal of contaminants and concentrates the proteins present in the complex. The purified proteins are then identified by mass spectrometry. In order to establish this methodology in our laboratory, this project proposes the use of N. crassa strains expressing the FLB-3 and RUV-1 fused to the PTP-tag (Protein C, TEV cleavage site and Protein a) for the purification of these complexes and identification of its protein components. RUV-1, a DNA helicase, participates in various complexes and it has also been studied in our laboratory, and therefore this protein will be used as a positive control for the standardization of the technique.