Expression Screening And Purificationanalysis Of E.coli Membrane Proteinbased On SfGFP

Membrane proteins have a variety of physiological functions,such as controlling the entry and exit of substances,signal and energy transmission,and so on,so they are widely used in drug target design and biological probe development.With the continuous update and development of biotechnology and biomedicine,the study of membrane proteins has attracted more and more attention from scholars.However,the low abundance and poor solubility of membrane proteins are two major problems that hinder the study of membrane protein structure and function.In this paper,based on the fusion expression of membrane protein and superfolder green fluorescent protein,the expression screening of eleven kinds of E.coli membrane proteins was carried out;the recombinant membrane protein with significantly increased expression was selected for small-scale expression optimization and large-scale cultivation of strains;Analysis and purification of recombinant membrane protein were carried out by nickel column affinity chromatography and size exclusion chromatography.Among the eleven E.coli membrane protein expression screening,the recombinant membrane protein PLSohB showed the best expression.After expression optimization experiments,the optimal expression conditions of PLSohB were determined: after second inoculation with TB liquid medium for three hours at 37°C,the E coli culture was mixed with IPTG(Isopropyl-?-D-thiogalactoside)that the final concentration is 1m M/L,and then induced and cultured at 30°C for five hours.A large amount of bacterial cells were cultivated under the optimal expression conditions,and the bacterial cells were broken and purified to obtain a protein sample with high purity.In addition,the size exclusion chromatogram result of membrane PLSohB verified the presumption that the membrane protein Soh B mainly exists in the form of octamer.In this experiment,the rapid screening of E.coli membrane protein expression through fusion expression overcomes the problem of unsatisfactory expression of membrane protein in a single mode,which is more convenient for subsequent purification and structural analysis and provides a new idea for membrane protein expression.