title: Separation of E. coli chaperonin groEL from β-galactosidase without denaturation.
abstract: Chaperonins are a class of ubiquitous proteins that assist and accelerate protein folding in the cell. The Escherichia coli groEL is the best known and forms a complex with its co-chaperonin groES in the presence of ATP and assists in the folding of nascent and misfolded substrate proteins. The purification of recombinant groEL results in a nearly homogeneous sample that consistently co-purifies with the major contaminant E. coli β-galactosidase. Removal of β-galactosidase using column chromatography alone is exceedingly difficult. This is due to the fact that the overall size, surface charge, isoelectric point and hydrophobicity of groEL and β-galactosidase are very similar. Therefore purification of groEL chaperonin to homogeneity requires denaturation of the complex into monomers with urea for separating the groEL from contaminating β-galactosidase followed by reassembly of the chaperonin complex. Here, we present a simple procedure for separating β-galactosidase along with many other impurities from groEL preparations under non-denaturing conditions. The groEL is first salted out with 50% ammonium sulfate. This step also precipitates β-galactosidase but this is then salted out by the addition of magnesium chloride which leaves groEL in solution. All remaining contaminants are removed by column chromatography.
authors.affiliation: Department of Chemistry, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA.
authors.forename: Sudheer K
authors.initials: SK
authors.lastname: Molugu
authors.affiliation: Department of Chemistry, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA.
authors.forename: Jihui
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Chemistry, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA. Electronic address: rbernal@utep.edu.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Bernal
mesh_terms: D018834:Chaperonin 60 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D011489:Protein Denaturation; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D001616:beta-Galactosidase
keywords: Chaperonin purification; GroEL; Protein salting out; β-Galactosidase