Structural and functional study of reconstituted peripheral benzodiazepine receptor
Structural and functional study of reconstituted peripheral benzodiazepine receptor
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DOI：
10.1006/bbrc.2001.4975
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发表时间：
2001-06-08
影响因子：
3.1
通讯作者：
Vidic, B
中科院分区：
文献类型：
--
作者：
Lacapère, JJ;Delavoie, F;Vidic, B
重组小鼠18 kDa外周型苯二氮卓受体（PBR）蛋白在大肠杆菌中过表达，并使用His Bind金属螯合树脂进行分离。重组PER蛋白用十二烷基硫酸钠纯化，并使用Bio-Beads SM 2作为去污剂重新掺入脂质体中。通过电子显微镜检查，重组PER样品的负染色显示形成了脂蛋白体。这些蛋白脂质体的冷冻断裂揭示了平均尺寸为3.5 +/- 0.25 nm的跨膜颗粒的存在，与重组PER蛋白的单体形式的存在一致。重构的蛋白质表现出以纳摩尔亲和力结合PER药物配体异喹啉甲酰胺PK 11195和胆固醇的能力。这些数据表明PER单体是结合药物配体和胆固醇的最小功能单元。(C)北京：科学出版社.
Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His Bind metal chelation resin. Recombinant PER protein was purified with sodium dodecyl sulfate and reincorporated into Liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PER samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PER protein. The reconstituted protein exhibited the ability to bind both the PER drug Ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PER monomer is the minimal functional unit, binding drug ligands and cholesterol. (C) 2001 Academic Press.