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Bourgeois_57301200_2018.pdf
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- Abstract
- The UPF0016 family is composed of membrane proteins found in all eukaryotes and many prokaryotes. Those proteins show conservation through evolution and are characterized by the presence of one or two copies of a consensus sequence E-Φ-G-D-(KR)-(ST) in which Φ can be any hydrophobic residue. Phenotypic assays and in vivo transport assays carried out in yeast have led to the hypothesis that those proteins can act as calcium, manganese and proton transporters. The major aim of this project was to purify a member of the UPF0016 family. For this purpose, three different members of the family have been selected: PfGdt1p from Pyrococcus furiosus, TgGdt1p from Thermococcus gammatolerans and DmGdt1p from Desulfovibrio magneticus. The genes have been cloned in pBAD plasmid under the control of an arabinose inducible promoter. The first step was the optimization of the production of each protein in E. coli. The three proteins were produced in E. coli and the best conditions were selected for the in vivo transport assays. These assays indicated that PfGdt1p and DmGdt1p transported calcium and manganese in a more efficient way than TgGdt1p. These two proteins were subsequently solubilized into different detergents. The detergents presenting the highest rate of solubilization for each protein (dodecylphosphocholine for DmGdt1p and n-dodecyl-β-D-maltoside for PfGdt1p) were selected. We developed a procedure to purify DmGdt1p and PfGdt1p. However, the amount of purified proteins is not sufficient to go further to reconstitution into liposomes and structural analysis. Therefore, our protocol still needs to be improved in order to obtain higher quantities of purified proteins.