Optimization of expression and the purification by organic extraction of the integral membrane protein EmrE.
EmrE is a member of the small multidrug resistance family of proteins and functions as a efflux transporter of lipophilic cations. This integral membrane protein is composed of 110 amino acids and is very hydrophobic with small loops exposed to the aqueous environment. This protein has been purified in a variety of ways including extraction with chloroform:methanol mixtures. This study explored culture growth and induction conditions, the parameters of organic solvent extraction, running conditions of a lipophilic column for lipid removal, as well as solubilization conditions. Optimal expression and purification protocols are crucial to the characterization goals of this protein. The experiments presented here led to an improvement in the yield of pure EmrE obtained by organic solvent extraction methods at a level of 0.9+/-0.2mg/L of culture. This is on the order of a 60% improvement over previous reports.
D017920:Antiporters / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D001692:Biological Transport; D002725:Chloroform; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D012997:Solvents / Q000737:chemistry*
D017920:Antiporters; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012997:Solvents; C075436:EmrE protein, E coli; D002725:Chloroform; D005998:Glycine; C100184:tricine