The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone-mediated refolding of heat-denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large-scale refolding processes. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1078-1085, 2009