摘要
目的:利用X线衍射技术解析孕烷X受体(PXR)配体结合结构域(LBD)蛋白晶体的3维结构。方法:对PXR蛋白LBD(130~434氨基酸残基)序列进行密码子优化并化学合成后克隆至pRSFDuet-1表达载体,再将载体导入大肠杆菌BL21(DE3),对PXR-LBD蛋白进行原核表达与分离纯化;采用晶体筛选试剂盒筛选蛋白结晶条件,采用悬滴法获得目标蛋白的晶体;对获得的蛋白晶体进行X线晶体衍射检测,并收集相关数据建立PXR-LBD的三维结构。结果:获得了PXR-LBD的高质量晶体并利用X线衍射解析了该蛋白质晶体的结构数据,使用Phenix.refine软件和COOT软件等对结构进行修正,最终获得了高分辨率的3维结构数据。结论:完成了孕烷X受体配体结合结构域蛋白晶体的X线衍射结构解析,为研究和开发PXR相关药物奠定了基础。
Objective: To obtain the three dimension structure of ligand binding domain(LBD) of pregnane X receptor(PXR) via protein crystals X-ray diffraction methods. Methods: The gene sequence of PXR-LBD(130~434 amino acid residues) was optimized and synthesized, and cloned into vector pRSFDuet-1. Next, the vector was transfected into E.coli BL21(DE3) to express PXR-LBD protein, and then the protein was isolated and purified.Protein crystallization conditions were screened by a crystal screening kit. Crystals of the target protein were obtained by drop method and subjected to X-ray crystal diffraction detection, and related data were collected to establish a three-dimensional structure of PXR-LBD. Results: The high-quality crystal of PXR-LBD was obtained.The high-resolution 3-dimensional structural data were acquired through X-ray diffraction analysis and Phenix.refine software and COOT software modification. Conclusion: The X-ray diffraction structure analysis of the PXRLBD protein crystals was successfully carried out, which laid a solid foundation for the research and development of PXR-related drugs.
引文
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