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Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination

DOI: 10.1016/j.ymeth.2011.08.015 DOI Help

Authors: Mitsunori Shiroishi (Japan Science and Technology Agency) , Takuya Yurugi-kobayashi (Japan Science and Technology Agency) , Satoshi Ogasawara (Kyoto University) , Hirokazu Tsujimoto (Japan Science and Technology Agency) , Chiyo Ikeda-suno (Japan Science and Technology Agency) , So Iwata (Diamond Light Source; Kyoto University; Imperial College, London) , Tatsuro Shimamura (Kyoto University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Methods , VOL 55 , PAGES 281 - 286

State: Published (Approved)
Published: December 2011

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 H1 receptor (H1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H1R 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.

Journal Keywords: GPCR; Expression; Pichia pastoris; Yeast; Structure; Membrane protein

Subject Areas: Medicine

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Added On: 01/02/2016 15:49

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