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 aggreg ates extensively in the cytoplasm of Escherichia coli, was used to sys tematically investigate the effects of heat-shock protein (hsp) overpr oduction 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'-bet a-galactosidase over a wide range of growth temperatures (30-42 degree s C), an increase in the concentration of the GroEL and GroES chaperon ins had a significant effect at 30 degrees C only, Coimmunoprecipitati on experiments confirmed that preS2-S'-beta-galactosidase formed a sta ble complex with DnaK, but not with GroEL, at 42 degrees C, When the i ntracellular concentration of chromosomal heat-shock proteins was incr eased by overproduction of the heat-shock transcription factor sigma(3 2), or by addition of 3% ethanol (v/v) to the growth medium, a 2-3-fol d 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 protei ns or specific chaperone operons did not significantly affect the synt hesis rates or stability of preS2-S'-beta-galactosidase and did not le ad to the disaggregation of preformed inclusion bodies. Rather, the im provements in the recovery of soluble and active fusion protein result ed primarily from facilitated folding and assembly. Our findings sugge st that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.