Purification of GroEL from an overproducing E. coli strain.

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Molecular chaperones are a class of abundant and ubiquitous proteins, which assist and accelerate protein folding in the cell. Classification of these proteins is continually changing owing to advances in our understanding of how these molecules function. One major group, the chaperonins (1), are defined as large, multimeric proteins that assemble into a double ring (or “double-doughnut”) structure. The crystal structure of the chaperonin GroEL from Escherichia coli (2) showed the 14 subunits arranged in two rings of 7 subunits. Each subunit has three distinct domains, the equatorial domain (through which the rings are joined), a hinge region (which facilitates movement), and the apical domain (where the unfolded protein substrate binds). There are many models for the mechanism of action of this molecule (for recent reviews, see 3), however it is generally accepted that the unfolded protein binds to the apical domain and is sequestered inside the cavity of the chaperonin owing to the binding of the cochaperonin, GroES, to the same sites on the apical domains. Release of the protein from the chaperonin occurs after ATP hydrolysis. Chaperonins are broadly further classified into two other groups based on source and sequence comparisons: Type I, which comprises bacterial, mitochondrial, and chloroplast chaperonins, and Type II, which comprises the archaeal and eukaryotic cytoplasmic chaperonins. All chaperonins have a subunit size of approx 60 kDa. GroEL has become the most examined and standard chaperonin in the field of chaperone-mediated protein folding. This chapter describes a purification protocol by which it is possible to obtain pure GroEL from an overproducing strain of E. coli. Several such strains have been produced in many laboratories and new constructs of GroEL, and its homologs can be generated easily using standard molecular biology techniques. Two main properties of the active chaperonin are exploited during its purification, that of its low isoelectric point (pI = 4.7) and large size (14×57 kDa).