In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model β-galactosidase fusion protein VP1LAC produced in DnaK - cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK+ cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK- background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli β-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK. © 2005 Wiley Periodicals, Inc.
- Protein folding
- Recombinant protein