Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor
Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor
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DOI：
10.1021/bi101159s
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发表时间：
2010-09-14
期刊：
影响因子：
2.9
通讯作者：
Rudolph, Rainer
中科院分区：
文献类型：
--
作者：
Schroeder-Tittmann, Kathrin;Bosse-Doenecke, Eva;Rudolph, Rainer
配体结合后胰高血糖素样肽 1 受体 (GLP-1R) 的激活导致胰腺细胞释放胰岛素。这种严格依赖葡萄糖的过程使得受体及其配体可用于治疗 II 型糖尿病。为了能够进行体外生物物理表征，我们将人全长 GLP-1R 在大肠杆菌胞浆中表达为包涵体。纯化后，使用人工伴侣系统将 SDS 与去污剂 Brij78 进行交换，从而实现 SDS 溶解的受体的重折叠。远紫外圆二色性光谱研究表明，该受体在 Brij78 胶束中采用特征性 α 螺旋结构。通过荧光猝灭和表面等离子体共振光谱对复性蛋白质的配体结合进行定量。在存在 Brij 胶束的情况下，重折叠受体以可逆一步机制结合激动剂 exendin-4，表观解离常数约为 100 nM。为了证明检测到的配体结合活性不仅归因于自主功能的 N 末端结构域 (nGLP-1R)，而且还归因于与近膜部分的额外接触，我们根据为全长 GLP-1R 建立的相同方案单独表达和重折叠胞外结构域。为了支持建议的多结构域结合模式，nGLP-1R 以较低的亲和力（K-app 在微摩尔范围内）和不同的动力学机制结合 exendin-4。 nGLP-1R 较低的配体亲和力完全是由于受体-配体复合物的动力学稳定性降低所致，与全长 GLP-1R 相比，nGLP-1R 的解离速度快 40 倍。总之，开发了一个框架，通过在大肠杆菌中重组表达来生产功能性人全长 GLP-1R，作为最终结构确定和包括蛋白质变体在内的严格生物物理表征的先决条件。
Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K-app in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is similar to 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants.