Membrane Protein Expression and Analysis in Yeast

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

“…An alternative with respect to shaking flasks is represented by the use of a bioreactor which enables tight control of oxygenation, pH, and temperature, leading to an increase of the production of the target protein as in the case of SLC35A1 [ 158 , 178 ]. Numerous publications of guides and protocols highlight the importance assigned to Pichia pastoris in the production of membrane proteins among which are transporters [ 31 , 32 , 179 ], even though the choice of this yeast is linked to its ability to efficiently secrete correctly folded heterologous proteins which facilitate purification and downstream processing such as reconstitution studies in proteoliposomes [ 30 , 58 , 180 , 181 ].…”

“…Moreover, the localization of an expressed membrane transporter in the yeast membrane can facilitate its recovery in a folded state [ 30 ]. Indeed, in this milieu, the human membrane proteins can find a more similar phospholipid/sterol composition with respect to the bacterial hosts [ 31 ]; furthermore, the possibility of increasing culture volume to obtain large quantities of folded protein [ 31 , 32 ] can be exploited either for functional or structural studies [ 20 , 33 , 34 ]. Indeed, yeast and especially P. pastoris can reach extremely high biomass, thus obviating the need to achieve extremely high levels of overexpression that could result in the aggregation and denaturation of membrane proteins.…”

A Simple Enrichment Procedure Improves Detection of Membrane Proteins by Immunoblotting

“…This could be done by centrifugation in a regular benchtop microcentrifuge. For example, a membrane fraction suitable for analysis by immunoblotting can be obtained from plant material by centrifugation at 21000 g for 2 h (2) or from yeast cell lysates by centrifugation at 16000 g for 45 min (3). We hypothesized that increasing the salt concentration during cell disruption may protect Pho87 from degradation and stimulate membrane precipitation, thus facilitating Pho87 enrichment by centrifugation.…”