Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

Abstract

Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins (IMPs), for various applications. This chapter details a series of protocols that progress towards the production of IMPs, their extraction and purification in presence of detergents, and their eventual reconstitution in lipid nanoparticles. These P. pastoris-oriented basic procedures can be further optimized in order to deliver IMP samples compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides a number of general guidelines, technical hints and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian IMPs.

Plant cellulose synthase membrane protein isolation directly fromPichia pastorisprotoplasts, liposome reconstitution, and its enzymatic characterization

“…Membrane proteins (MP) are highly amphipathic, temperature, and shear-sensitive. Conventional methods of membrane protein extraction, such as bead-beating, sonication, product entrapment, and homogenization, exert a lot of physical pressure on the cells that could be detrimental to the membrane protein integrity [28][29][30]. Moreover, methods like homogenization might also extract the MPs that are folded incorrectly and not processed completely since it involves the usage of whole-cell lysate [25].…”

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 ].…”

Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses

“…It also relies on a number of other parameters, such as detergent and protein concentrations, ionic strength, addition of co-factors and stabilizing compounds and finally temperature and incubation time. The optimal condition is most generally protein-dependent and should ideally be screened and adjusted for each MP of interest (30,(43)(44)(45). The extraction procedure presented here with DM as a detergent has been optimized for hCTR1.…”

“…Once MPs are solubilized, a panel of strategies and techniques may be considered to purify them to homogeneity, mainly depending on the biochemical properties of the protein itself, on the affinity tags it may present, and on the composition of the buffer it has been solubilized in (30,46). Here we describe a two-step purification protocol specifically designed for hCTR1.…”