嗜热脂肪芽胞杆菌色氨酰-tRNA合成酶BsTrpRS的表达、纯化及其与创新霉素复合物的单晶制备和X-射线衍射分析  

Prokaryotic expression and purification of tryptophanyl-tRNA synthetase from Baccillus stearothermophilus and preliminary crystallographic study on its complex with chuagnxinmycin

作　　者：樊帅[1] 吕广新 金媛媛[1] 杨兆勇[1] FAN Shuai;LYU Guang-xin;JIN Yuan-yuan;YANG Zhao-yong(Department of Microbial Pathway Engineering,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Chinese Academy of Medical Sciences,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室,北京100050

出　　处：《中国医药生物技术》2021年第3期222-228,共7页Chinese Medicinal Biotechnology

基　　金：中国医学科学院医学与健康科技创新工程项目(2019-I2M-1-005);国家自然科学基金面上项目(81872782)。

摘　　要：目的获得来源于嗜热脂肪芽胞杆菌色氨酰-tRNA合成酶(BsTrpRS)与创新霉素复合物的晶体结构,为创新霉素的理性设计提供结构基础。方法利用大肠杆菌表达系统异源表达获得BsTrpRS可溶性表达,采用亲和层析色谱、离子交换色谱和分子排阻色谱纯化目的蛋白,以期获得纯度在90%以上的BsTrpRS,通过等温滴定量热法检测BsTrpRS与创新霉素的亲和力。选用悬滴-水蒸汽扩散法筛选蛋白质结晶条件,通过优化结晶条件获得高质量晶体,利用上海光源蛋白质晶体学光束线站收集X-ray衍射数据。结果运用分子生物学方法成功构建含bstrprs基因的重组质粒,转化BL21(DE3)得到重组表达菌株。运用亲和层析、离子交换色谱和分子排阻色谱等蛋白纯化技术,获得高纯度BsTrpRS蛋白。利用ITC技术确证创新霉素与BsTrpRS之间亲和力为3.2μmol/L。通过晶体筛选优化,最佳结晶条件为1.8 mol/L K_(2)HPO_(4),pH 7.6,37℃,0.75%(v/v)1,3-丙二醇,最终获得X-ray衍射分辨率为2.06A的BsTrpRS/创新霉素二元复合物晶体,其晶胞参数为a=91.675A,b=91.675A,c=152.37A,α=90.000°,β=90.000°,γ=120.000°,晶体的马修斯系数为2.48A^(3)/Da,最小非对称单位内含有2个蛋白分子,溶剂百分比为50.46%。结论创新霉素与BsTrpRS复合物晶体结构的获得,有望对创新霉素小分子结构优化及设计提供指导方向及重要依据。Objective Solving the crystal structure of Tryptophanyl-tRNA Synthetase(BsTrpRS)from Baccillus stearothermophilus in complex with chuangxinmycin will provide us more insight into the function of chuangxinmycin in physiology and pharmacology,and also help us design and optimize the chuangxinmycin-based drugs.Methods The soluble expression of BsTrpRS protein was obtained by Escherichia coli expression system.The target protein was purified by affinity chromatography,ion exchange chromatography and molecular size exclusion chromatography in order to obtain BsTrpRS,with purity of more than 90%.The binding affinity between BsTrpRS and chuangxinmycin was evaluated using isothermal titration calorimetry analysis.High quality crystal for X-ray diffraction was obtained on the protein crystallography beamline(BL18U1)at the Shanghai Synchrotron Radiation Facility by employing vapor diffusion suspension method.Results The recombinant plasmid containing bstrprs gene was successfully constructed by molecular biological method,and the strain BL21(DE3)with high expression of BsTrpRS protein was obtained by optimization.High purity Bs Trp RS protein was obtained by affinity chromatography,ion exchange chromatography and molecular size exclusion chromatography.The binding affinity of the BsTrpRS/chuangxinmycin was 3.2μmol/L.The complex crystals of BsTrpRS and chuangxinmycin was obtained at optimum conditions(1.8 mol/L K_(2)HPO_(4),0.75%v/v 1,3-propylene glycol,pH 7.6,37℃),and the resolution of diffraction data up to 2.06A.The Matthews coefficient of the crystal is 2.48A^(3)/Da,which corresponds to nearly 50.46%solvent content with two subunits of BsTrpRS protein in the asymmetric unit.Conclusion The crystal structure of BsTrpRS with chuangxinmycin is expected to provide guidance and basis for the optimization and design of small molecular analogues of chuangxinmycin.

关 键 词：创新霉素 色氨酰-tRNA合成酶 共结晶 X-射线衍射 

分 类 号：R915[医药卫生—微生物与生化药学]