title: Purification of the human G protein-coupled receptor adenosine A(2a)R in a stable and functional form expressed in Pichia pastoris.
abstract: The isolation of membrane proteins with the aim of producing highly pure, homogeneous, stable, and functional material remains challenging, and it is often necessary to develop protein-specific purification protocols by trial and error. One key tool that is required in the development of a suitable protocol is a functional assay. This unit describes a range of different protocols for isolation of the human adenosine A2a receptor (A(2a)R). These protocols show the importance of developing a robust method for comparing the quality of protein obtained by a combination of biophysical analyses including SDS-PAGE, analytical size-exclusion chromatography, and functional analysis. One of the keys to isolating and maintaining a functional receptor, found not only in the optimal protocol described here but in other published examples, is that there should be no more than two chromatographic steps.
authors.affiliation: Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom.
authors.forename: Shweta
authors.initials: S
authors.lastname: Singh
authors.affiliation: Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom.
authors.forename: Minghao
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bertheleme
authors.affiliation: School of Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Strange
authors.affiliation: Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom.
authors.forename: Bernadette
authors.initials: B
authors.lastname: Byrne
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010843:Pichia / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D059748:Proteolysis; D043705:Receptor, Adenosine A2A / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
chemical_list: D008565:Membrane Proteins; D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins