Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins.

PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.