Production and purification of GFP-tagged Gdt1p, a secondary cation transporter from S. cerevisiae, using native nanodiscs

Details

Supervisors

Morsomme, Pierre ; Gaiffe, Alexandra

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Congenital Disorders of Glycosylation (CDG) represent a class of rare human diseases impacting the pathway of protein glycosylation. Affected patients suffer from numerous symptoms as growth and psychomotor retardation, muscular weakness and abnormal fat distribution. Ten years ago, various mutations in the gene encoding the Golgi-localized protein TMEM165 were associated with this inherited pathology. This protein belongs to the well conserved GDT1 protein family, characterized by the presence of one or two copies of the consensus pattern: Glu-φ-Gly-Asp-(Arg/Lys)-(Ser/Thr) (φ representing any hydrophobic residues). Many researches helped to better understand the role of proteins belonging to this family. They revealed the influence of protein members in Ca2+, Mn2+ and probably protons homeostasis. However, many critical information are still missing such as in the mechanism and the direction of transport. Structural analyses based on the S. cerevisiae protein, Gdt1p, could lead to a better comprehension of the transport of GDT1 protein family members. The aim of this project was to purify the yeast ortholog, Gdt1p, embedded in native nanodiscs. To perform affinity purification trials, different tags were added to Gdt1p. The plasmid containing the tagged version of Gdt1p (10His-GFP-TEV-Δ23Gdt1p) was obtained based on the fusion of two DNA fragments by the Gibson assembly method before being transformed into the bacterium Lactococcus lactis. GFP-tagged proteins were extracted from cells by cell lysis method and ultracentrifugation steps were then achieved to isolate membrane proteins from soluble proteins. These protein extracts were then analyzed by western blot. Results suggested a degradation happening between tags and Gdt1p. However, its origin was unclear. To counteract in vitro degradation, different cell lysis methods (bead mills, cryogenic grinding) were tested whereas, to prevent in vivo degradation, changes in the cell culture temperature were performed. In the meantime, in vivo transport assays using the sensitive Fura-2 probe were performed and indicated the ability of GFP-tagged Gdt1p to transport calcium ions across the bacterial membrane. Since the protein was functional and expressed, membrane proteins were solubilized with styrene-maleic acid (SMA) polymers and purified by affinity chromatography. Finally, this first purification attempt turned out not to be successful because of solubilization and binding issues. Despite those poor results, several tracks could be considered for optimizing Gdt1p production and purification.