Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris

被引：14
作者：
Shukla, Arun Kumar
Haase, Winfried
Reinhart, Christoph
Michel, Hartmut
机构：
[1] Max Planck Inst Biophys, Dept Mol Membrane Biol, D-60438 Frankfurt, Germany
[2] Max Planck Inst Biophys, Dept Biol Struct, D-60438 Frankfurt, Germany
来源：
PROTEIN EXPRESSION AND PURIFICATION | 2007年 / 55卷 / 01期
关键词：
GPCR; bradykinin receptor; heterologous expression; glycosylation; localization;
D O I：
10.1016/j.pep.2007.02.021
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
The human bradykinin subtype 2 receptor (B2R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5 pmol/mg of total membrane protein. [H-3]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (K-d = 0.87 +/- 0.1 nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B2R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B2R suggested that the recombinant receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the recombinant receptor for structural studies. (C) 2007 Elsevier Inc. All rights reserved.
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页码：1 / 8
页数：8
相关论文
共 42 条
[1]   Increased AT1 receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness [J].
AbdAlla, S ;
Lother, H ;
el Massiery, A ;
Quitterer, U .
NATURE MEDICINE, 2001, 7 (09) :1003-1009
[2]   Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen [J].
André, N ;
Cherouati, N ;
Prual, C ;
Steffan, T ;
Zeder-Lutz, G ;
Magnin, T ;
Pattus, F ;
Michel, H ;
Wagner, R ;
Reinhart, C .
PROTEIN SCIENCE, 2006, 15 (05) :1115-1126
[3]   Regulation of cardiovascular signaling by kinins and products of similar converting enzyme systems -: Downregulation of bradykinin B2 receptor in human fibroblasts during prolonged agonist exposure [J].
Blaukat, A ;
Micke, P ;
Kalatskaya, I ;
Faussner, A ;
Müller-Esterl, W .
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, 2003, 284 (06) :H1909-H1916
[4]   Molecular tinkering of G protein-coupled receptors: an evolutionary success [J].
Bockaert, J ;
Pin, JP .
EMBO JOURNAL, 1999, 18 (07) :1723-1729
[5]  
CRONAN JE, 1990, J BIOL CHEM, V265, P10327
[6]   Human vascular kinin receptors of the B-2 type characterized by radioligand binding [J].
Gessi, S ;
Rizzi, A ;
Calo, G ;
Agnello, G ;
Jorizzo, G ;
Mollica, G ;
Borea, PA ;
Regoli, D .
BRITISH JOURNAL OF PHARMACOLOGY, 1997, 122 (07) :1450-1454
[7]   Production of the human D2S receptor in the methylotrophic yeast P-pastoris [J].
Grünewald, S ;
Haase, W ;
Molsberger, E ;
Michel, H ;
Reiländer, H .
RECEPTORS & CHANNELS, 2004, 10 (01) :37-50
[8]   BRADYKININ RECEPTORS - PHARMACOLOGICAL PROPERTIES AND BIOLOGICAL ROLES [J].
HALL, JM .
PHARMACOLOGY & THERAPEUTICS, 1992, 56 (02) :131-190
[9]   Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris [J].
Kim, TK ;
Zhang, RD ;
Feng, W ;
Cai, H ;
Pierce, W ;
Song, ZH .
PROTEIN EXPRESSION AND PURIFICATION, 2005, 40 (01) :60-70
[10]  
Klabunde T, 2002, CHEMBIOCHEM, V3, P929, DOI 10.1002/1439-7633(20021004)3:10<928::AID-CBIC928>3.0.CO
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