基于BLI技术的BamAβ-barrel结合化合物检测方法的建立  

Establishment of a method to detect the binding activity of compounds and BamAβ-barrel based on BLI assay

作　　者：郑怡凡 朱小红 陈明华 卢宇 魏元娟 时文静 司书毅 李妍 Zheng Yifan;Zhu Xiaohong;Chen Minghua;Lu yu;Wei Yuanjuan;Shi Wenjing;Si Shuyi;Li yan(Beijing Key Laboratory of Antimicrobial Agents,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050)

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、国家新药(微生物)筛选实验室,北京100050

出　　处：《中国抗生素杂志》2024年第4期448-456,共9页Chinese Journal of Antibiotics

基　　金：国家自然基金(No.32141003);中国医学科学院医学与健康科技创新工程(No.2021-I2M-1-070)。

摘　　要：目的以革兰阴性菌外膜蛋白折叠辅助因子关键蛋白BamA为靶蛋白,基于生物膜干涉(Biolayer interferometry,BLI)技术建立化合物与BamA蛋白β折叠结构域(BamA_(β-barrel))结合活性的评价方法,为建立靶向BamA蛋白的抗革兰阴性菌先导物奠定基础。方法应用BLI方法检测BamA_(β-barrel)与已知的阳性化合物darobactin的结合活性。原核表达并纯化带有His标签的大肠埃希菌BamA_(β-barrel)蛋白,使用表面活性剂LDAO对其进行复性和折叠;使用生物素标记折叠和未折叠蛋白,并结合到超级链霉亲和素(super streptavidin,SSA)生物传感器,然后检测蛋白与不同浓度的darobactin结合信号的变化,同时做无蛋白或darobactin稀释液对照;空白对照采用未结合生物素化的BamA_(β-barrel)蛋白的传感器,检测上述系列稀释样品。相应信号采用Steady state analysis方式拟合分析,计算平衡常数(KD)值。结果成功获得高纯度的折叠状态BamA_(β-barrel)蛋白,通过BLI技术检测到折叠状态的BamA_(β-barrel)与阳性化合物darobactin具有良好结合活性且呈现浓度依赖性,R^(2)为0.9998,KD值为(2.2E-06±8.0E-08)M。结论基于BLI技术成功建立了折叠状态的BamA_(β-barrel)-化合物结合活性的评价方法,为后续BamA蛋白靶向性抗革兰阴性菌抗生素的发现建立基础。Objective Using the key protein BamA of the Gram-negative bacterial outer membrane protein folding cofactor as the target protein,this study established a method to detect the binding activity between BamA_(β-barrel)and compounds based on biolayer interferometry(BLI)technology and laid the foundation for screening antibiotics against Gram-negative bacteria targeting the BamA protein.Methods Double subtraction BLI was used to detect the interaction of BamA_(β-barrel)and the positive compound darobactin.E.coli BamA_(β-barrel)was expressed and purified and then it was refolded with LDAO in vitro.The refolded BamA_(β-barrel)or unfolded BamA_(β-barrel)were labeled with biotin and bound to the Super Streptavidin(SSA)biosensor.The binding of darobactin to BamA_(β-barrel)was detected based on the changes in binding signals.To identify the diluted samples,a blank control was employed,which involved employing unbound biotinylated BamA_(β-barrel)protein sensors.This control was performed simultaneously with the protein-free or darobactin dilution control.The signal that corresponded to it was fitted and evaluated using steadystate analysis in order to get the value of the equilibrium constant(KD).Results High-purity folded BamA_(β-barrel)was successfully obtained.The folded BamA_(β-barrel)bound to the sensor showed good binding activity with darobactin with R^(2)=0.9998 and KD=2.2E-06±8.0E-08 M.Conclusion An optimized BLI method for detecting the interaction between compounds and BamA_(β-barrel)had been established,which could be used for antibiotics against Gram-negative bacteria targeting BamA_(β-barrel).

关 键 词：革兰阴性菌 Darobactin BamAβ-barrel 生物膜干涉技术 

分 类 号：R978.1[医药卫生—药品]