Characterization of an engineered water-soluble variant of the full-length human mu opioid receptor
Characterization of an engineered water-soluble variant of the full-length human mu opioid receptor
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DOI：
10.1080/07391102.2019.1677502
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发表时间：
2019-10-16
影响因子：
4.4
通讯作者：
Liu，Renyu
中科院分区：
文献类型：
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作者：
Xi，Jin;Xiao，Jie;Liu，Renyu
人MU阿片受体跨膜区的一种水溶性变体(wsMOR-TM)此前已被鉴定。本研究探索了工程水溶性受体(wsMOR-FL)的全长版本是否可以在大肠杆菌中过表达，以及它是否保持了水的溶解性、结合能力和热稳定性。WsMOR高表达，且纯度较高。ColiBL21(DE3)细胞(EMD/Novagen)，正如我们之前为wsMOR-TM报道的那样。天然的N末端和C末端都被添加回高度工程的wsMOR-TM。为了纯化目的，在N端增加了6个组氨酸标签。用原子力显微镜对wsMOR-FL的单体状态、二级结构的圆二色性和热稳定性进行了表征。测定了其与纳曲酮的结合力。与天然人MOR相比，wsMOR-FL具有与拮抗剂纳曲酮相似的螺旋二级结构含量和相似的亲和力(NM)。该受体的二级结构在广泛的pH范围内(6-9)保持稳定。与跨膜部分相比，全长受体的二级结构能耐受较宽的温度范围(10-90 °C)。用原子力显微镜直接成像时，受体主要以单体形式存在于溶液中。本研究证明，人Mu受体的功能性全长水溶变异体可以用ANE过表达和纯化。结肠杆菌表达系统。这为研究人MOR的结构和功能特性提供了一种新的工具。N-末端和C-末端增强了蛋白质在这种特定的水溶性变体中的热稳定性。
A water-soluble variant of the transmembrane domain of the human mu opioid receptor (wsMOR-TM) was previously characterized. This study explored whether the full-length version of the engineered water-soluble receptor, (wsMOR-FL), could be overexpressed inEscherichia coliand if it would retain water solubility, binding capability and thermostability. wsMOR was over-expressed and purified inE. coliBL21(DE3) cells (EMD/Novagen) as we reported previously for the wsMOR-TM. Both native N and C termini were added back to the highly engineered wsMOR-TM. Six His-tag was added in the N terminus for purification purposes. The wsMOR-FL was characterized using atomic force microscope for its monomeric state, circular dichroism for its secondary structure and thermostability. Its binding with naltrexone is also determined. Compared to the native human MOR, wsMOR-FL displays similar helical secondary structure content and comparable affinity (nM) for the antagonist naltrexone. The secondary structure of the receptor remains stable within a wide range of pH (6–9). In contrast to the transmembrane portion, the secondary structure of full-length receptor tolerated a wide range of temperature (10–90 °C). The receptor remains predominantly as a monomer in solution, as directly imaged using atomic force microscopy. This study demonstrated that functional full-length water-soluble variant of human mu receptor can be over-expressed and purified using anE. coliover-expression system. This provides a novel tool for the investigation of structural and functional properties of the human MOR. N- and C-termini strengthened the thermostability of the protein in this specific water soluble variant.Communicated by Ramaswamy H. Sarma