Separation of E. coli chaperonin groEL from β-galactosidase without denaturation

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.