MOLECULAR CHAPERONES, FOLDING CATALYSTS, AND THE RECOVERY OF ACTIVE RECOMBINANT PROTEINS FROM ESCHERICHIA-COLI - TO FOLD OR TO REFOLD

The high-level expression of recombinant gene products in the gramnega tive bacterium Escherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular protea ses or its deposition into biologically inactive aggregates known as i nclusion bodies. It has recently become clear that in vivo protein fol ding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroE L-GroES systems, suppress off-pathway aggregation reactions and facili tate proper folding through ATP-coordinated cycles of binding and rele ase of folding intermediates. On the other hand, folding catalysts (fo ldases) accelerate rate-limiting steps along the protein folding pathw ay such as the cis/trans isomerization of peptidyl-prolyl bonds and th e formation and reshuffling of disulfide bridges. Manipulating the cyt oplasmic folding environment by increasing the intracellular concentra tion of all or specific folding modulators, or by inactivating genes e ncoding these proteins, holds great promise in facilitating the produc tion and purification of heterologous proteins. Purified folding modul ators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemicall y denatured polypeptides. This review examines the usefulness and limi tations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.