Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.
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TLDR
The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.Abstract:
Background: The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins. Results: A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold. Conclusion: The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.read more
Citations
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References
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Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein
TL;DR: Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine.
Journal ArticleDOI
Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR/O, the TetR/O and AraC/I1-I2 Regulatory Elements
Rolf Lutz,Hermann Bujard +1 more
TL;DR: Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced.
Journal ArticleDOI
Pathways of chaperone-mediated protein folding in the cytosol
TL;DR: In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding.
Journal ArticleDOI
DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.
TL;DR: The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.
Journal ArticleDOI
Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB
Axel Mogk,Toshifumi Tomoyasu,Pierre Goloubinoff,Stefan G.D. Rüdiger,Daniel Röder,Hanno Langen,Bernd Bukau +6 more
TL;DR: The data indicate that large‐sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.
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