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Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.

de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B - BMC Biotechnol. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBL Heidelberg, Heidelberg, Germany. ario.demarco@ifom-ieo-campus.it

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.

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Chaperone overproduction upon IPTG induction in E. coli cells carrying plasmid combinations 1 to 6. Lysates of non-induced cells (U) and IPTG-induced cells (I) grown overnight at 20°C were separated by SDS-PAGE and Coomassie-stained. The various plasmid combinations present in the tested cells are indicated.
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Figure 2: Chaperone overproduction upon IPTG induction in E. coli cells carrying plasmid combinations 1 to 6. Lysates of non-induced cells (U) and IPTG-induced cells (I) grown overnight at 20°C were separated by SDS-PAGE and Coomassie-stained. The various plasmid combinations present in the tested cells are indicated.

Mentions: Host cell were transformed with plasmids in 5 combinations. Combination 1 (pBB530 and pBB535) for overproduction of KJE; combination 2 (pBB540 and pBB535) for overproduction of KJE and ClpB; combination 3 (pBB528 and pBB541) for overproduction of ELS; combination 4 (pBB540 and pBB542) for overproduction of KJE, ClpB and high amounts of ELS; combination 5 (pBB540 and 550) for overproduction of KJE with ClpB and lower amounts of ELS (Fig. 1). Continuous growth of these cells in medium containing 100 μM IPTG resulted in an increase of DnaK (18–22 fold), ClpB (15–18 fold), GroEL (30 fold, combination 4; 5 fold, combination 5) over wild type levels (Fig. 2), without causing apparent growth defects (data not shown). Host cells containing different combinations of these plasmids were subsequently transformed with plasmids expressing 50 different recombinant genes of prokaryotic and eukaryotic origin (Table 1) from IPTG-controlled promoters. The encoded proteins include monomers and oligomers, cytosolic, membrane bound and secreted proteins, full-length, fragmented and fused proteins (fusion to GST, Trx, DsbA or NusA) [see Additional file 1], with molecular weights ranging between 7.5 and 118 kDa. Proteins were hexa-histidine tagged to allow affinity purification of the soluble fractions.


Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.

de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B - BMC Biotechnol. (2007)

Chaperone overproduction upon IPTG induction in E. coli cells carrying plasmid combinations 1 to 6. Lysates of non-induced cells (U) and IPTG-induced cells (I) grown overnight at 20°C were separated by SDS-PAGE and Coomassie-stained. The various plasmid combinations present in the tested cells are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904446&req=5

Figure 2: Chaperone overproduction upon IPTG induction in E. coli cells carrying plasmid combinations 1 to 6. Lysates of non-induced cells (U) and IPTG-induced cells (I) grown overnight at 20°C were separated by SDS-PAGE and Coomassie-stained. The various plasmid combinations present in the tested cells are indicated.
Mentions: Host cell were transformed with plasmids in 5 combinations. Combination 1 (pBB530 and pBB535) for overproduction of KJE; combination 2 (pBB540 and pBB535) for overproduction of KJE and ClpB; combination 3 (pBB528 and pBB541) for overproduction of ELS; combination 4 (pBB540 and pBB542) for overproduction of KJE, ClpB and high amounts of ELS; combination 5 (pBB540 and 550) for overproduction of KJE with ClpB and lower amounts of ELS (Fig. 1). Continuous growth of these cells in medium containing 100 μM IPTG resulted in an increase of DnaK (18–22 fold), ClpB (15–18 fold), GroEL (30 fold, combination 4; 5 fold, combination 5) over wild type levels (Fig. 2), without causing apparent growth defects (data not shown). Host cells containing different combinations of these plasmids were subsequently transformed with plasmids expressing 50 different recombinant genes of prokaryotic and eukaryotic origin (Table 1) from IPTG-controlled promoters. The encoded proteins include monomers and oligomers, cytosolic, membrane bound and secreted proteins, full-length, fragmented and fused proteins (fusion to GST, Trx, DsbA or NusA) [see Additional file 1], with molecular weights ranging between 7.5 and 118 kDa. Proteins were hexa-histidine tagged to allow affinity purification of the soluble fractions.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBL Heidelberg, Heidelberg, Germany. ario.demarco@ifom-ieo-campus.it

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.

Show MeSH