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Separation of E. Coli Chaperonin groEL From β-galactosidase Without Denaturation

Separation of E. Coli Chaperonin groEL From β-galactosidase Without Denaturation

Sudheer K Molugu, Jihui Li, Ricardo A Bernal

J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Dec 15;1007:93-9.

PMID: 26590880

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9031112-C	β-Galactosidase, E. coli		9031-11-2	Price
IAR4241040	Chaperonin 60 from Escherichia coli			Price

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