UDP-3-O-(R-羟基十四酰)-N-乙酰氨基葡萄糖脱乙酰基酶的表达、纯化及与ACHN-975复合物的X-射线衍射分析  

Prokaryotic expression and purification of UDP-3-O-(R-hydroxymyristoyl)-N-acetylglucosamine deacetylase from Aquifex aeolicus and the preliminary crystallographic study on its complex with ACHN-975

作　　者：李丹阳[1] 樊帅[1] 金媛媛[1] 杨兆勇[1] LI Dan-yang;FAN Shuai;JIN Yuan-yuan;YANG Zhao-yong(Department of Microbial Pathway Engineering, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China)

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所微生物代谢工程研究室

出　　处：《中国医药生物技术》2019年第5期406-411,共6页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金面上项目(81872782);中国医学科学院医学与健康科技创新工程项目(2016-I2M-3-022)

摘　　要：目的以 UDP-3-O-(R-羟基十四酰)-N-乙酰氨基葡萄糖脱乙酰基酶(LpxC)-ACHN-975 晶体复合物结构为基础,研究小分子与靶蛋白的结合位点与方式,进而依据复合物结构设计该化合物,以获得活性强、毒性低的药物小分子。方法运用大肠杆菌表达系统对超嗜热菌(Aquifex aeolicus)来源的 AaLpxC 进行异源表达,采用 Zn^2+-NTA 亲和层析、Q-HP 强阴离子交换色谱和 Superose 12 分子排阻色谱对重组蛋白进行分离纯化,对 AaLpxC 进行结构生物学研究,采用蒸汽扩散-悬滴法进行结晶条件筛选以及优化。结果获得纯度在 90%以上的超嗜热菌来源的 AaLpxC,经过结晶条件的优化获得分辨率为 1.21 А(1 А= 1 × 10^-10 m)的 AaLpxC 与 ACHN-975 的复合物晶体的衍射数据,其晶胞参数为 a = 65.569 А,b = 65.569 А,c = 131.595 А,α= 90.000°,β= 90.000°,γ= 120.000°。结论 ACHN-975 与 AaLpxC 复合物晶体结构的获得有望对 ACHN-975 小分子结构优化及设计提供指导方向及重要依据。Objective Based on the complex structure of ACHN-975 with LpxC and the study on the corresponding binding site and binding model. It is promising to design and optimize ACHN-975 analogues to obtain optimized molecules with strong activity and low toxicity. Methods The heterogeneous expression system of Escherichia coli was used to express AaLpxC from Aquifex aeolicus. The recombinant protein was purified by Zn^2+-NTA affinity chromatography, Q-HP strong anion exchange and Superpose 12 exclusion, with the final purity being over 90%. The crystallization condition was screened and optimized by hanging-drop vapor diffusion method, and the crystal structure of AaLpxC with ACHN-975 was studied. Results The complex crystals of AaLpxC and ACHN-975 were obtained at optimum conditions (14 mg/ml, 100 mmol/L HEPES, pH 7.0, 6% PEG8000, 10 % glycol, 15 ℃), and the resolution of diffraction data was up to 1.21 А(1 А= 1 × 10^-10 m). The lattice parameters of crystal (a = 65.569 А, b = 65.569 А, c = 131.595 А,α= 90.000°,β= 90.000°,γ= 120.000°) was obtained. Conclusion The obtained crystal structure of LpxC with ACHN-975 is expected to provide guidance and basis for the optimization and design of small molecular analogues of ACHN-975.

关 键 词：LpxC ACHN-975 蛋白纯化 结晶 X-射线衍射 

分 类 号：R91[医药卫生—药学]