<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>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 <jats:italic>E. coli</jats:italic> cytosol on the solubility of recombinant proteins.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>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 <jats:italic>de novo</jats:italic> folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins <jats:italic>in vivo</jats:italic>. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>The engineered <jats:italic>E. coli</jats:italic> 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.</jats:p> </jats:sec>