Folding problems of overproduced client proteins can be caused by limitations in the chaperone capacity of the host cells. The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. coli, IbpA and IbpB (IbpAB), intercalate into protein aggregates and thereby facilitate the KJE/ClpB dependent disaggregation and refolding. Moreover, the small heat shock proteins (sHSP) of E. KJE furthermore cooperates with ClpB to solubilize aggregated proteins and refold them to the native state in a concerted action with ELS. due to thermal denaturation or intrinsic instability. KJE and ELS also prevent aggregation and promote refolding of preexisting proteins which lost their native conformation e.g. coli cytosol, the folding of newly synthesized proteins is assisted by the ribosome-associated Trigger Factor, the DnaK system (DnaK with its DnaJ and GrpE cochaperones KJE), and the GroEL system (GroEL with its GroES cochaperone ELS). 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.Ĭhaperones assist the folding of newly synthesized proteins to the native state and provide a quality control system that refolds misfolded and aggregated proteins. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. 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. ResultsĪ two-step procedure was found to show the strongest enhancement of solubility. coli cytosol on the solubility of recombinant proteins. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful.

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