The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of d-glyceraldehyde-3-phosphate dehydrogenase~GAPDH! in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either longor short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced aLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.

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Reduced denatured lysozyme tends to aggregate at neutral pH and competition between productive folding and aggregation substantially reduces the efficiency of refolding. Trigger factor, a folding catalyst and chaperone can, depending on the concentration of trigger factor and the solution conditions, cause either a substantial increase (chaperone activity) or a substantial decrease (antichaperone activity) in the recovery of native lysozyme as compared with spontaneous refolding. When trigger factor is working as a chaperone, the reactivation rates of lysozyme are decelerated and aggregation decreases with increasing trigger factor concentrations. Under conditions where antichaperone activity of trigger factor dominates, the reactivation rates of lysozyme are accelerated and aggregation is increased. Trigger factor and lysozyme were both released from the aggregates on re-solubilization with urea indicating that trigger factor participates directly in aggregate formation and is incorporated into the aggregates. The apparently dual effect of trigger factor toward refolding of lysozyme is a consequence of the peptide binding ability and may be important in regulation of protein biosynthesis.