Expression And Purification Of Membrane Protein Complex Of Active Transport Ton System In Escherichia Coli

Membrane proteins undertake a variety of physiological activities in basic cell functions,and are also the main types of drug targets.The study of membrane protein structural biology can promote the development of vaccines,new drugs,and ecology.At present,the main bottleneck of membrane protein research is the difficulty in obtaining a large number of high-purity membrane protein samples.The research object of this article is Ton complex,which is a pathway protein used by Gram-negative bacteria to take in external nutrients,including membrane proteins ExbB,ExbD and TonB.exbB and exb D are on the same gene cluster.The Ton system is very important for the nutrient uptake and metabolism of Gram-negative bacteria,but the stoichiometry,structural characteristics and mechanism action of the protein in the Ton system are not yet clear.The purpose of this study is to obtain the most abundant ExbB membrane protein,ExbB-ExbD subunit complex,and the complete complex ExbB-ExbDTonB of the Ton system through expression and purification,which can be used for subsequent research on the structure and function of the system.Firstly,the recombinant vector pLX001 was constructed based on the fusion expression strategy of sf GFP.The expression conditions and purification were optimized according to the color label of sf GFP.The ExbB protein samples were obtained by expression,purification and TEV enzyme cutting.Secondly,the co-expression vector pLX002 was constructed to express ExbBD subunit complex,and the co-expression vector pLX003 was constructed on the basis of pLX002 to express ExbBD-TonB complex with complete Ton system.Considering that ExbD protein has two copies in Ton complex,in the presence of wild-type ExbD,the purified Ton complex has two different forms(r-ExbD2,r ExbD-wt ExbD),which will interfere with subsequent non-denaturing mass spectrometry experiments.Therefore,the Escherichia coli knockout strain BL21(DE3)-ΔexbBD was constructed by CRISPR / Cas9 system.The co-expression vectors pLX002 and pLX003 were transformed into the knockout strain for expression and purification to obtain the ExbBD subunit complex and ExbBD-TonB complex samples,which laid the foundation for the structural analysis and functional experiment of Ton system.