Production and purification of a GFP-tagged version of Gdt1p, a secondary ion transporter from S. cerevisiae

Details

Supervisors

Morsomme, Pierre ; Stribny, Jiri

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries

Abstract

Congenital Disorders of Glycosylation (CDG) are a rapidly growing family of rare inherited diseases causing numerous severe symptoms such as muscular weaknesses, skeletal abnormalities, psychomotor retardation, etc. Among these disorders, TMEM165-CDG has been identified in 2012. This disease results from specific mutations in the gene coding for the Golgi-localized TMEM165 transporter. This protein belongs to the very well conserved Uncharacterized Protein Family 0016 defined by the presence of a consensus motif (one or two copies) consisting of the following residues: E-Φ-G-D-(KR)-(ST), Φ indicating any hydrophobic residue. Recent findings revealed that members of this family are involved in calcium, manganese and very likely pH homeostasis. However, some critical points are still missing regarding the biochemistry and molecular functions of this family. Due to the lack of purified proteins in an active form, those specific areas remain unclear whereas it could lead to a better knowledge of the physiological role of these transporters. In this work, we proposed a purification strategy focusing on the optimized version of GFP-Gdt1p fusion protein, Gdt1p being the S. cerevisiae ortholog from the UPF0016 family. A tagged version – 10His-GFP-TEV-Gdt1p – was first constructed to allow purification by the IMAC method. Then, subcellular localization and analyses of total protein extracts showed partial in vivo degradation of the recombinant protein. Protein abundance was therefore optimized by testing different expression systems. The production was finally maximized by using S. cerevisiae pep4∆ strain under control of the strong pTPI1 promoter. Solubilization of the recombinant protein was then optimized in the mild DDM detergent before performing a purification trial. Despite the need for further optimizations, this first purification attempt proved to be very promising, showing for the first time a purified UPF0016 member produced by S. cerevisiae detected by a Coomassie blue staining. Following purification improvements, functional analyses by protein reconstitution into proteoliposomes would represent a major step towards a better knowledge of the UPF0016 protein family.