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(BamAβ-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BamAβ-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BamAβ-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BamAβ-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BamAβ-barrel蛋白,通过BLI技术检测到折叠状态的BamAβ-barrel与阳性化合物darobactin具有良好结合活性且呈现浓度依赖性,R2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BamAβ-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.
Establishment of a method to detect the binding activity of compounds and BamAβ-barrel based on BLI assay
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 BamAβ-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 BamAβ-barrel and the positive compound darobactin.E.coli BamAβ-barrel was expressed and purified and then it was refolded with LDAO in vitro.The refolded BamAβ-barrel or unfolded BamAβ-barrel were labeled with biotin and bound to the Super Streptavidin(SSA)biosensor.The binding of darobactin to BamAβ-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 BamAβ-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 steady-state analysis in order to get the value of the equilibrium constant(KD).Results High-purity folded BamAβ-barrel was successfully obtained.The folded BamAβ-barrel bound to the sensor showed good binding activity with darobactin with R2=0.9998 and KD=2.2E-06±8.0E-08 M.Conclusion An optimized BLI method for detecting the interaction between compounds and Bam Aβ-barrel had been established,which could be used for antibiotics against Gram-negative bacteria targeting BamAβ-barrel.
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