An improved method for the expression screening of membrane protein-GFP fusions in yeast

Abstract

The expression and purification of membrane proteins is an extremely challenging area of work within Protein Science. Membrane proteins are required for compound screening and structure determination in industry. Here we describe some new and innovative methodology in developing the membrane protein GFP fusion primary expression screening in yeast. This methodology enables the expression of membrane proteins fused to GFP in both Saccharomyces cerevisiae and Pichia pastoris systems. This capability helps facilitate screening of constructs to establish which are suitable for membrane protein production for compound screening and structure determination\n\nIn terms of the primary screening work, we have developed both agar plate and liquid plate expression methodology in yeast. The two approaches correspond well, but the agar plate method is more rapid and we have shown it to have the advantage of allowing cells to be taken directly into confocal microscopy for immediate cell localisation data. Innovative work to extend the methanol induction time in the Pichia agar plate method established good differentiation from the background. A novel agar plate method was also developed for S.cerevisiae which is also presented. These screening methods allow triaging of constructs for either membrane protein preps for biochemical assays or progression to fluorescence size exclusion chromatography; where various detergents can be screened to determine the most appropriate for membrane protein solubilisation, the starting point for purification, crystallisation and structure determination.\n\nMembrane targets depicted to demonstrate the improved primary screening methodology are a copper transporter Ctr1p from S.cerevisiae and a water transporter Aqp4 from human origin.\n\nHighlightsAn improved method for the production of recombinant MP-GFP fusions in yeast is presented using agar plates.\n\nAn agar plate method for MP-GFP expression screening is described for Pichia pastoris, with improved induction methodology by the simple addition of methanol, allowing longer induction times for expression clarity.\n\nA new simple rapid agar plate method for MP-GFP expression screening is described for Saccharomyces cerevisiae.\n\nCells can be taken directly from agar plates into confocal microscopy studies for immediate cell localisation data and triaging.\n\nLiquid plate based screening methods are also described for both yeasts in comparison, to show there is corresponding data, helping validate the new agar plate methods.