基于BLI技术的脂多糖转运蛋白LptA/LptC相互作用检测方法的建立

Development of a BLI assay-based method for detecting LptA/LptC interaction

摘要:

革兰阴性菌脂多糖转运蛋白 (Lipopolysaccharide transport，Lpt) LptA和LptC通过稳定的相互作用对脂多糖装配起到重要作用，它们相互作用的阻断会导致脂多糖层缺损和菌体死亡，因此具备成为抗菌药物筛选靶标的可行性。文中应用生物膜干涉 (Biolayer interferometry，BLI) 技术对LptA/LptC相互作用进行检测，为建立体外的LptA/LptC蛋白相互作用阻断剂筛选方法奠定基础。首先在大肠杆菌Escherichia coli BL21(DE3) 中进行大肠杆菌LptA全长、LptA去信号肽和LptC蛋白的表达；纯化的蛋白使用生物素标记后结合到预先稀释液封闭的超级链霉亲和素 (Super streptavidin，SSA) 生物传感器，然后再检测与未标记蛋白之间的结合信号，同时做无蛋白的稀释液对照；使用同样的方法检测生物素化蛋白与小分子的结合以及相互作用的阻断；空白对照采用未结合生物素化蛋白的传感器，检测上述系列稀释样品。响应信号采用稳态分析 (Steady state analysis) 方式拟合，计算样品平衡常数 (KD) 值。本研究成功获得高纯度的LptA和LptC蛋白，并且检测到结合传感器的LptC蛋白与LptA全长蛋白和LptA去信号肽蛋白均具有良好的结合活性，KD值分别为2.9e–7±7.9e–8、6.0e–7±2.8e–8；结合传感器的LptA去信号肽蛋白与LptC蛋白具有良好的结合活性，KD值为9.6e–7±7.2e–9；所有结合曲线呈现出明显的快结合-快解离形态。小分子化合物IMB-881能够与LptA结合来阻断LptA/LptC之间的相互作用，与LptC之间无结合活性。文中首次建立了基于BLI技术的LptA/LptC相互作用检测方法，并且证实该方法能够用于小分子阻断剂阻断活性的评价，为后续的LptA/LptC蛋白相互作用阻断剂筛选奠定了基础。

Abstract:

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e–7±7.9e–8 and 6.0e–7±2.8e–8, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e–7±7.2e–9. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.