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Journal of Biochemistry Advance Access published online on October 18, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj211
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© 2006 The Japanese Biochemical Society.
Received October 10, 2006
Accepted October 13, 2006

Regular Paper

Purification of Human {beta}2-Adrenergic Receptor Expressed in Methylotrophic Yeast Pichia pastoris

Shuji Noguchi 1 and Yoshinori Satow 1 *

1 Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

* To whom correspondence should be addressed.
Yoshinori Satow, E-mail: satowy{at}mol.f.u-tokyo.ac.jp


  Abstract

Human {beta}2-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/l-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.

Keywords: {beta}2-adrenergic receptor; G-protein coupled receptor; membrane protein; Pichia pastoris; structural biology.
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