Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli
Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli
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DOI：
10.1093/jb/mvm213
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发表时间：
2008-02-01
影响因子：
2.7
通讯作者：
Machida, Sachiko
中科院分区：
文献类型：
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作者：
Kumano-Kuramochi, Miyuki;Xie, Qiuhong;Machida, Sachiko
晚期糖基化终产物受体（RAGE）是一种参与糖尿病并发症发展的多配体受体。利用大肠杆菌表达系统，我们成功地表达和纯化了人RAGE的c端生物素化胞外结构域（hsRAGE），该结构域由三个携带三个假定的二硫键的免疫球蛋白样结构域组成。超过90%的hsRAGE以可溶性形式在trxB和g5突变型大肠杆菌Origami （DE3）中表达。大多数hsRAGE被c端AviTag生物素化，并在未经任何处理的情况下通过链亲和素稳定地固定在基质上。未糖基化的固定化hsRAGE识别其配体，如AGEs。生物素化的hsRAGE也能够像酶联免疫分析法中使用的抗体一样用于微滴孔中AGEs的检测。SPR分析表明，RAGE对AGE-BSA的解离常数（K-d）在两态反应模型下为23.1 nM，在1:1结合模型下为13.5 nM，与细胞表面的RAGE相当。这些结果表明，生物素化的hsRAGE不仅可用于分析age -配体相互作用，还可用于检测age。
The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K-d) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.