Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids  

作　　者：Yuqin Wang Pingping Fan Shanyu Zhang Shuanghong Yan Shuo Huang 

机构地区：[1]State Key Laboratory of Analytical Chemistry for Life Sciences,School of Chemistry and Chemical Engineering,Nanjing University,Nanjing 210023,China [2]Chemistry and Biomedicine Innovation Center(ChemBIC),Nanjing University,Nanjing 210023,China

出　　处：《Biophysics Reports》2021年第5期355-364,共10页生物物理学报（英文版）

基　　金：This project was funded by National Natural Science Foundation of China(31972917,91753108 and 21675083);the Fundamental Research Funds for the Central Universities(020514380257 and 020514380261);programs for high-level entrepreneurial and innovative talents introduction of Jiangsu Province(individual and group program),Natural Science Foundation of Jiangsu Province(BK20200009);Excellent Research Program of Nanjing University(ZYJH004);Shanghai Municipal Science and Technology Major Project,State Key Laboratory of Analytical Chemistry for Life Science(5431ZZXM1902);Technology innovation fund program of Nanjing University,China Postdoctoral Science Foundation(2021M691508).

摘　　要：Mycobacterium smegmatis 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.

关 键 词：MspA nanopore Prokaryotic expression Affinity purification Single molecule sensing 

分 类 号：Q503[生物学—生物化学]