Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human 渭-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

详细信息    查看全文

• 作者：M貌nica Rosa ; Joan Josep Bech-Serra ; Francesc Canals ; Jean Marie Zajac ; Franck Talmont ; Gemma Arsequell ; Gregorio Valencia
• 刊名：Journal of Proteome Research
• 出版年：2015
• 出版时间：August 7, 2015
• 年：2015
• 卷：14
• 期：8
• 页码：3162-3173
• 全文大小：665K
• ISSN：1535-3907

文摘

Human 渭-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.