DIVERGENT EFFECTS OF CHAPERONE OVEREXPRESSION AND ETHANOL SUPPLEMENTATION ON INCLUSION-BODY FORMATION IN RECOMBINANT ESCHERICHIA-COLI

The proper folding of aggregation-prone recombinant proteins in Escher ichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or oth er agents that upregulate the synthesis of all heat-shock proteins (hs ps), We have investigated the effect of combining direct chaperone ove rproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, add ition of 3% (v/v) ethanol to the growth medium prior to inoculation im proved the chaperone-mediated increase in the yields of active preS2-S '-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzy matic activity were 5-fold higher relative to control cells and preS2- S'-beta-galactosidase aggregation was almost entirely abolished, Combi ning DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in act ivity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was rai sed, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert. In contras t, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'- beta-galactosidase. Our results show that ethanol can exert complex an d divergent effects on inclusion body formation and that the beneficia l effect of the solvent on recombinant protein folding cannot simply b e explained by an increase in the intracellular concentration of molec ular chaperones. (C) 1997 Academic Press.