The quantitative similarity between protein activity in the soluble cell fraction and that of the aggregated forms of both enzymes and fluorescent proteins (Table 1) demonstrates that physiological aggregation as inclusion bodies does not necessarily split protein population into active and inactive fractions. Probably, protein solubility (observed as the occurrence in the soluble cell fraction) does not necessarily indicate the acquisition of a correctly folded and thus active structure. In this context, soluble micro-aggregates have been described  and recently characterized in detail . The non complete coincidence between solubility and folding has been previously indicated by exhaustive mutational analysis of model proteins , showing that the genetic determinants of protein aggregation and misfolding are not coincident. In this way, natively unfolded proteins are unstructured but soluble . Therefore, determinations of GFP-fusions solubility by using fluorescence as reporter  could have eventually been indicative of folding-misfolding extend rather than solubility-insolubility, since inclusion bodies formed by GFP fusions can be highly fluorescent (Figure 2). Furthermore, solubility does not appear to be an all-or-nothing attribute and polypeptide chains might exhibit a continuum of folding states in both soluble and insoluble cell fractions, between which they are dynamically transferred with the assistance of cellular folding modulators . In this context, the occurrence and evolution of 'soluble' aggregates in bacteria (namely misfolded species occurring in the soluble cell fraction and presumably inactive)  could explain the variable specific activity observed in the soluble cell fraction of bacteria producing recombinant β-galactosidases .
Inversely, our results prove a major occurrence of native or native-like protein in inclusion bodies. In fact, deposition as inclusion bodies might even result in the enrichment of active species as suggested by the specific activity (166 % of that found in the soluble cell fraction; Table 1) and catalytic properties (Figure 3A) of VP1LAC inclusion bodies. This observation can be then again indirectly indicative of the presence of enzymatically inactive protein in the soluble cell fraction, since protein deposition is not expected to favour a correct folding.
Finally, although the existence of native-like structure in bacterial inclusion body proteins has been previously reported , here we demonstrate that this is not anecdotic but probably the architectonic nature of these kind of aggregates, as inclusion bodies formed by four structurally different proteins all display significantly high biological activity. Interestingly, the active and properly folded polypeptides in inclusion bodies coexist with a molecular β-sheet organization also manifest in all cases, although the extent of β-sheet structure and its coincidence with the biological activity of the aggregates cannot be quantitatively evaluated. Since is highly improbable that enzyme active sites involved in the intermolecular β-sheet structure could be themselves active, we suggest that enzymatic activity or fluorescence are supported by properly folded molecules or molecule segments. Aggregation, observed as protein deposition driven by intermolecular interactions between solvent-exposed hydrophobic patches  would not necessarily disturb the conformation of all protein domains, and the active site would be still functional if misfolded, aggregation-prone regions are located in a distant site of the polypeptide chain. Alternatively, properly folded and active molecules could coexist with β-sheet-enriched (inactive) versions of the same species, and both situations could in fact take place simultaneously in single aggregate units. Further structural and functional analysis would hopefully solve this issue.
From an applied point of view, inclusion bodies, being formed by sequence-specific interaction between homologous protein patches result in highly pure protein microparticles . Since they are also porous and highly hydrated , efficient substrate diffusion would probably occur for most of the (or at least many) biotechnologically relevant aggregated enzymes, thus opening the possibility for a new industrial market of enzymatically active inclusion bodies.