Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

Abstract

BackgroundVarious protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.ResultsThe ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.ConclusionCompared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Pichia pastoris as an expression host for membrane protein structural biology

“…Radioligand binding analysis also revealed that for several of the receptors higher functional expression was achieved in P. pastoris compared to the mammalian cell system [46 ]. Comparison of muscarinic acetylcholine receptor M2 expression in P. pastoris and Spodoptera frugipera insect cells revealed similar levels of specific activity and binding affinity but the higher cell densities achieved in P. pastoris meant that the overall yield in this system was twice that in the insect cells [47]. The most successful host for the recombinant production of eukaryotic membrane proteins for structural studies is currently the insect cell system [48], however P. pastoris is not far behind.…”

Properties of Human Tumor Suppressor 101F6 Protein as a Cytochrome b561 and Its Preliminary Crystallization Trials

“…Most importantly, Pichia pastoris cell is a very suitable host for the expression of membrane proteins, particularly for integral membrane proteins, because of its eukaryotic nature (e.g., presence of membranes within the cytosolic milieu). It was proposed that, compared to insect cells (e.g., Sf9 cell) or other mammalian cultured cells, Pichia pastoris cells are much easier to handle, can be grown at lower cost, and can be expressed quicker in a large scale (Asada et al, 2011). Such successful examples for expressing eukaryotic membrane proteins were reported previously (Weiß et al, 1998;Wetterholm et al, 2008;Nakanishi et al, 2009a;Nakanishi et al, 2009b;Alisio & Mueckler, 2010;Ostuni et al, 2010;Mizutani et al, 2011).…”

Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

“…(10) After solubilizing the membrane with digitonin/Na-cholate solution, M2-i3d bound to the high-affinity inverse agonist 3-quinuclidinyl-benzilate (QNB) was purified by using an aminobenztropine (ABT) affinity column and hydroxyapatite column, as previously described. (11) The eluate was concentrated with Amicon Ultra (Merck Millipore, Billerica, MA) and dialyzed against 20 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 5% glycerol, 0.05% n-dodecyl-b-D-maltopyranoside (DDM, Anatrace, Maumee, OH), and 0.01% cholesterol hemisuccinate (CHS, Sigma-Aldrich, St. Louis, MO).…”