Expression Purification And Oligomeric State Analysis Of MscL By Mass Spectrometry

The Mechanosensitive channel of Large conductance(MscL)is a kind of important membrane protein in the biological membrane ion channel.MscL acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis.Thelarge pore size,essential for function,requires the formation of oligomers with tetramers,pentamers or hexamers observed depending on the species and experimental approach.This research is mainly about the oligomeric state of MscL.In the first step,MscL gene is inserted into E.coli pET-28 vector.Then,the MscL gene is induced to express MscL protein.After that MscL peotein is purified and analysed by mass spectrometry.The membrane protein is fat soluble protein and difficult to crystallized,so in the purification and analysis of MscL,detergent DDM is added.Nondenaturing mass spectrometry is highly sensitive and the oligomer state of MscL can be determined.To improve expression efficiency of the protein and obtain better mass spectromrtry signal,fussion expression of MscL with GFP(green fluorescence protein)and ThrxA is part of the work.Overall,studies of MscL have provided significant insight to the field,and serve as a paradigm for the analysis of non-homologous,eukaryotic mechanosensitive channel proteins.