Wang Yuqin, Fan Pingping, Zhang Shanyu, Yan Shuanghong, Huang Shuo
State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing 210023, China.
Biophys Rep. 2021 Oct 31;7(5):355-364. doi: 10.52601/bpr.2021.210016.
porin A (MspA), possessing a short (0.6 nm) and narrow (1.2 nm) constriction that enables its high spatial resolution of sensing, has emerged as an optimum choice for nanopore sequencing and nano-reactive sensing. However, prepared MspA nanopores cannot be obtained from any commercial vendors. We here provide a highly simplified protocol for MspA preparation. The protocol yields ~10 mg fully oligomerized protein per liter of culture and is straightforward to follow. With the prepared MspA nanopores, discrimination of immobilized ssDNA oligonucleotides can be performed, which serves as a demo application to demonstrate core procedures of single molecule sensing using MspA. This method is also in principle compatible with all other MspA mutants, which might merit the need to develop a variety of MspA based nano-reactive sensors.
孔蛋白A（MspA）具有短（约0.6纳米）且窄（约1.2纳米）的收缩结构，使其具有高空间分辨率的传感能力，已成为纳米孔测序和纳米反应传感的最佳选择。然而，市售渠道无法获得制备好的MspA纳米孔。我们在此提供一种高度简化的MspA制备方案。该方案每升培养物可产生约10毫克完全寡聚化的蛋白质，且易于遵循。利用制备好的MspA纳米孔，可以对固定化的单链DNA寡核苷酸进行区分，这作为一个演示应用，展示了使用MspA进行单分子传感的核心程序。该方法原则上也与所有其他MspA突变体兼容，这可能满足开发各种基于MspA的纳米反应传感器的需求。
