Production of the stable human histamine H 1 receptor in Pichia pastoris for structural determination

Mitsunori Shiroishi, Takuya Kobayashi, Satoshi Ogasawara, Hirokazu Tsujimoto, Chiyo Ikeda-Suno, So Iwata, Tatsuro Shimamura

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

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 1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H 1R 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.

Original languageEnglish
Pages (from-to)281-286
Number of pages6
JournalMethods
Volume55
Issue number4
DOIs
Publication statusPublished - Dec 1 2011

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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