Abstract
BackgroundThe molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide.ResultsWhile thermal denaturation results in the formation of heterogeneous aggregates that are rather stable in composition, protein deposition as inclusion bodies renders homogenous but strongly evolving structures, which are progressively enriched in the main protein species while gaining native-like structure. Although both type of aggregates display common features, inclusion body formation but not thermal-induced aggregation involves deposition of functional polypeptides that confer biological activity to such particles, at expenses of the average conformational quality of the protein population remaining in the soluble cell fraction. In absence of DnaK, however, the activity and conformational nativeness of inclusion body proteins are dramatically impaired while the soluble protein version gains specific activity.ConclusionThe chaperone DnaK controls the fractioning of active protein between soluble and insoluble cell fractions in inclusion body-forming cells but not during thermally-driven protein aggregation. This cell protein, probably through diverse activities, is responsible for the occurrence and enrichment in inclusion bodies of native-like, functional polypeptides, that are much less represented in other kind of protein aggregates.
Highlights
The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities
It has been recently proposed that protein aggregation in bacteria is not an all-or-nothing process [16], since the quality of recombinant proteins extends over a continuum of conformational forms [17], that include soluble aggregates [18,19] and active protein entrapped in true, refractile inclusion bodies [9,13]
We have explored the occurrence of active, properly folded polypeptides in inclusion bodies and in thermally driven aggregates formed by the same protein species, and the influence of the main chaperones DnaK and GroEL in the quality of the deposited polypeptides and of those remaining in the soluble fraction
Summary
The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. Bacterial inclusion bodies are dynamic structures, they grow resulting from an unbalanced equilibrium between constant protein deposition and removal that is lost in absence of protein synthesis [4,7]. They contain significant amounts of protein in a native-like form [8,9,10,11,12], a fact that is reflected by the important extent of biological activity exhibited by inclusion bodies formed by very different target proteins [1315]. The conformational status of the inclusion body protein is influenced, among others, by environmental factors such as the growth temperature [20] and the gene expression strategy [21], but little is known about the role of cellular factors on the quality of protein species in both soluble and insoluble cell fractions
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