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Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both L L-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of L L-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process. ß

The molecular organisation of protein aggregates, formed under physiological conditions, has been explored by in vitro trypsin treatment and electron microscopy analysis of bacterially produced inclusion bodies (IBs). The kinetic modelling of protein digestion has revealed variable proteolysis rates during protease exposure that are not compatible with a surfacerestricted erosion of body particles but with a hyper-surfaced disintegration by selective enzymatic attack. In addition, differently resistant species of the IB proteins coexist within the particles, with half-lives that differ among them up to 50-fold. During in vivo protein incorporation throughout IB growth, a progressive increase of proteolytic resistance in all these species is observed, indicative of folding transitions and dynamic reorganisations of the body structure. Both the heterogeneity of the folding state and the time-dependent folding transitions undergone by the aggregated polypeptides indicate that IBs are not mere deposits of collapsed, inert molecules but plastic reservoirs of misfolded proteins that would allow, at least up to a certain extent, their in vivo recovery and transference to the soluble cell fraction.z 2000 Federation of European Biochemical Societies.

By immunostaining and transmission electron microscopy, chaperones DnaK and GroEL have been identified at the solvent-exposed surface of bacterial inclusion bodies and entrapped within these aggregates, respectively. Functional implications of this distinct localization are discussed in the context of Escherichia coli protein quality control.