Purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris.
Human beta2-adrenergic receptor is a G-protein-coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. N-terminal histidine-tagged gene constructs with optimized codon usage were designed so as to obtain Pichia pastoris transformants with a high expression level. The constructs were inserted into the pPIC9 vector, and then electroporated into the SMD1168 strains. The highest expression level obtained was about 4 mg/liter-culture broth. The dissociation constant of the receptor in the membrane fraction was 1.2 nM toward CGP-12177 antagonist. The receptor was solubilized with sucrose monolaurate and purified with a series of chromatography steps including anion-exchange, Ni-Sepharose, alprenolol-Agarose, and hydroxyapatite columns. The receptor was heterogeneously glycosylated, showing broad SDS-PAGE bands around 70-90 kDa. After endoglycosidase treatment, the receptor appeared as a single band around 45 kDa, and was further purified with hydroxyapatite and gel-filtration columns. The receptor was eluted as a sharp peak at the gel-filtration elution volume corresponding to a molecular mass of 117 kDa. The saccharide-trimmed receptor thus purified is homogeneous as analyzed with SDS-PAGE, shows the dissociation constant of 4.7 nM toward CGP-12177 antagonist, and is suitable for crystallization experiments.
Noguchi
Shuji
S
Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033.
Satow
Yoshinori
Y
D002850:Chromatography, Gel; D018274:Electroporation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000378:metabolism*; D011412:Propanolamines / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism
D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C033394:CGP 12177; D000432:Methanol