Fiche publication
Date publication
janvier 2017
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr WAGNER Renaud
Tous les auteurs :
Hartmann L, Metzger E, Ottelard N, Wagner R
Lien Pubmed
Résumé
In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favoring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography.
Mots clés
Chromatography, Affinity, Chromatography, Gel, Membrane Proteins, chemistry, Pichia, cytology, Protein Conformation, Protein Engineering, Protein Folding, Protoplasts, metabolism, Recombinant Proteins, chemistry
Référence
Methods Mol. Biol.. 2017 ;1635:45-56
