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Abstract: The human bradykinin subtype 2 receptor (B2R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5pmol/mg of total membrane protein. [3H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (K d =0.87±0.1nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B2R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B2R suggested that the recombinant receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the recombinant receptor for structural studies. [Copyright &y& Elsevier]

Abstract: One of the major objectives of the Membrane Protein Network program (MePNet, www.mepnet.org) is to express one hundred G-protein-coupled receptors (GPCRs) in the yeast Pichia pastoris. We have developed an antibody-based assay in order to select for the best behaving clones at each step of the receptor preparation, from expression to solubilization. This assay allowed us to quantify the expression of Flag-tagged GPCRs present in various sample types, from crude P. pastoris extracts to native membrane preparations and detergent solubilised fractions. It combines the specificity of ELISA, the sensitivity of enhanced chemiluminescence detection and the speed of high-throughput screening, and can detect as low as 0.001% (w/w) flag-tagged recombinant receptor present in a crude extract. The method was applied to sort recombinant GPCR clones, to rank receptor expression levels and to screen for detergent solubilization efficiency using the human β2-adrenergic receptor expressed in P. pastoris as a benchmarking standard. [Copyright &y& Elsevier]

Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins. • P. pastoris , a promising expression system for ATP-dependent transport proteins. • Sequential use of different mild detergents resulted in high yields of target proteins. • Hydrophilic IMAC matrices essential for monodisperse, active transport proteins. • Low-resolution cryo-EM density maps of NEO1-p and YPK9-p were generated. • P-, N- and A-domains of NEO1-p and YPK9-p identified by comparing with homologs. [ABSTRACT FROM AUTHOR]

Abstract: Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields. [Copyright &y& Elsevier]