G蛋白偶联受体
重组DNA
HEK 293细胞
表面等离子共振
配体结合分析
蛋白质工程
计算生物学
化学
生物化学
转染
兴奋剂
生物
受体
分子生物学
纳米技术
酶
基因
纳米颗粒
材料科学
作者
Thao T. Ho,Jasmine Nguyen,Juping Liu,Paweł Stańczak,Allison Thompson,Yingzhuo Yan,Jasmine Chen,C.K. Allerston,Charles L. Dillard,Xu Hu,Nicholas J. Shoger,Jill S. Cameron,Mark E. Massari,K. Aertgeerts
标识
DOI:10.1016/j.pep.2017.03.002
摘要
Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay.
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