title: Production of the stable human histamine H₁ receptor in Pichia pastoris for structural determination.
abstract: G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.
authors: Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors: Mitsunori
authors: M
authors: Shiroishi
authors: Takuya
authors: T
authors: Kobayashi
authors: Satoshi
authors: S
authors: Ogasawara
authors: Hirokazu
authors: H
authors: Tsujimoto
authors: Chiyo
authors: C
authors: Ikeda-Suno
authors: So
authors: S
authors: Iwata
authors: Tatsuro
authors: T
authors: Shimamura
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D046508:Culture Techniques; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D014176:Protein Biosynthesis; D059748:Proteolysis; D011738:Pyrilamine / Q000737:chemistry; D011969:Receptors, Histamine H1 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae
chemical_list: D011969:Receptors, Histamine H1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011738:Pyrilamine