Divergent Genetic Control of Protein Solubility and Conformational Quality in Escherichia coli

Rehosting of Bacterial Chaperones for High-Quality Protein Production

“…2A). These results were in marked contrast to those of similar experiments with E. coli, where coexpression of DnaK and DnaJ with mGFP resulted in much lower fluorescence levels than in the control cells (10).…”

“…Misfolding-prone green fluorescent protein (GFP) fusions synthesized in DnaK knockouts (10) rendered higher protein yields but reduced recombinant protein solubility compared to those in wild-type cells. In contrast, the overexpression of dnaK and dnaJ genes in E. coli enhanced the proportion of soluble recombinant proteins by stimulating Lon-and ClpP-mediated proteolysis of aggregated proteins, reducing overall protein yields (10). Very recently, the molecular basis of the DnaKJmediated proteolytic enhancement has been solved by fine dissection of the interaction between DnaKJ and 32 .…”

“…Such conformational effect of the substrate seems to be the mechanistic platform of both foldase and disaggregase activities exhibited by DnaK/DnaJ on their substrates, including RepA and unfolded proteins (22). In recombinant E. coli cells, DnaK/ DnaJ could mediate conformational perturbations of misfolded proteins at the surface of inclusion bodies, where DnaK localizes (4), exposing them to the stress proteases Lon and ClpP (10) and promoting digestion during refolding attempts. Such a dual role of DnaK/DnaJ in stimulating protein folding but also enhancing degradation of protease-sensitive protein species could be the cause of the controversial data obtained from their use as folding modulators and the reason for the only transient rise of soluble protein species refolded in vivo from bacterial inclusion bodies (3).…”

“…coli, but that was not the case for the proteolytic stimulation of recombinant proteins, which is highly dependent on the E. coli proteases Lon and ClpP (Table 1) (10). Under the assumption that insect cell proteases would not recognize the target sites exposed by the activity of DnaKJ (no Lon and ClpP orthologs have so far been identified in insects), we rehosted the E. coli DnaKJ pair for coproduction along with recombinant protein by using the baculovirus expression system.…”

“…Under the assumption that insect cell proteases would not recognize the target sites exposed by the activity of DnaKJ (no Lon and ClpP orthologs have so far been identified in insects), we rehosted the E. coli DnaKJ pair for coproduction along with recombinant protein by using the baculovirus expression system. For these studies, we used a proteolytically unstable GFP (mGFP), already characterized for recombinant protein quality analysis in bacteria (10), and generated two transfer vectors designed to express mGFP either alone or together with the bacterial chaperones DnaK and DnaJ, using the vector pAcAB4 (2). mGFP and dnaJ genes were placed under the control of the p10 promoter, and dnaK was placed under the control of the polyhedrin promoter.…”