Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli
Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli
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DOI：
10.1016/j.pep.2006.07.019
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发表时间：
2007-02-01
影响因子：
1.6
通讯作者：
Ruoho, Arnold E.
中科院分区：
文献类型：
--
作者：
Ramachandran, Subramaniam;Lu, Hongliang;Ruoho, Arnold E.
σ受体曾经被认为是一类阿片受体，现在被认为是独特的孤儿受体，其特征在于结合各种药理学试剂的能力，如孕酮（类固醇）、氟哌啶醇（抗精神病药）和滥用药物如可卡因和甲基苯丙胺。σ-1受体是一种223个氨基酸的蛋白质，被认为具有两个跨膜区段。我们已经开发了一种纯化豚鼠sigma-1受体后，在大肠杆菌中过表达的麦芽糖结合蛋白（MBP）融合和提取与Triton X-100的计划。使用直链淀粉柱和Ni 2+亲和柱的亲和层析用于纯化σ-1受体。通过该方法纯化的σ-1受体是如通过SDS-PAGE评估的26 kDa多肽，以高亲和力结合σ配体，并且可以用σ-1受体光探针[I-125]-碘代叠氮可卡因特异性地光亲和标记。使用[H-3]-（+）-喷他佐辛的配体结合表明，Triton X-100中约一半的纯化蛋白与放射性配体结合。在-20 ℃下，在含有30%甘油的缓冲液中，0.5% triton中的MBP-σ-1受体和σ-1受体最大稳定约两周。(c)2006年爱思唯尔公司All rights reserved.
Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [I-125]-iodoazidococaine. Ligand binding using [H-3]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol. (c) 2006 Elsevier Inc. All rights reserved.