Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. To fold or to refold.
The high-level expression of recombinant gene products in the gram-negative bacterium Escherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as the cis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.
Thomas
J G
JG
University of Washington, Department of Chemical Engineering, Seattle 98195-1750, USA.
Ayling
A
A
Baneyx
F
F
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002384:Catalysis; D004926:Escherichia coli / Q000502:physiology*; D005818:Genetic Engineering; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D018832:Molecular Chaperones / Q000502:physiology*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins