Danko,
High pressures have been used in order to recover in solution protein aggregates (precipitates) .
Below I cite a reference and copy/paste some parts. Different amounts of pressure have different effects in the proteins but it seems that there are pressures that can fold, or denaturate or solubilize proteins.
So here you are:
Vol. 96, Issue 23, 13029-13033, November 9, 1999
Applied Biological Sciences
High pressure fosters protein refolding from aggregates at high concentrations
Richard J. St. John*, John F. Carpenter, and Theodore W. Randolph*,
High hydrostatic pressures (1-2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and -lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml. Inclusion bodies containing -lactamase could be refolded at high yields of active protein, even without added GdmHCl.
The purpose of this study is to investigate the use of high pressure as an alternative to high concentrations of strong chaotropes for protein disaggregation and refolding. Pressures between 1 and 3 kbar will reversibly dissociate oligomeric proteins into subunits (20-23). Pressures above 4 kbar begin to denature the secondary structure of proteins (24-26). Pressure has been shown to reduce aggregation rates during refolding from fully soluble, denatured protein (27). Also, aggregation of P22 tailspike protein was reduced from 41% to 18% by pressure treatment of a 100 µg/ml solution (28). However, pressure has not been used as a tool to obtain native protein from insoluble aggregates at relatively high protein concentrations, with high yields, nor from covalently crosslinked aggregates or inclusion bodies. We hypothesize that if the pressure effects on aggregates of non-native protein molecules are similar to those on native multimeric proteins, then there must exist a "pressure window" where pressure is high enough to solubilize aggregates, but still allow refolding to the native conformation. The model systems chosen to test this hypothesis were agitation-induced insoluble aggregates of recombinant human growth hormone (rhGH), chaotrope-induced aggregates of hen egg white lysozyme that were covalently crosslinked through non-native disulfides, and inclusion bodies containing -lactamase produced in Escherichia coli.
Remarkably, high pressure refolding of rhGH appears to be independent of protein concentration. Once optimal refolding conditions were determined, we increased protein concentrations up to 8.7 mg/ml, orders of magnitude higher than conditions typically used for refolding studies. Samples pressurized for 24 hr at 2 kbar, 1 M GdmHCl also achieved 100% recovery of rhGH from aggregates (data not shown).
We conclude that pressure provides a powerful tool for obtaining native protein molecules from insoluble aggregates, inclusion bodies, and even covalently crosslinked aggregates. This process allows proteins to be refolded from such aggregates at concentration orders of magnitude greater than reported previously and at yields approaching 100%. In fact, recovery of both rhGH and lysozyme exhibited independence of protein concentration in the ranges studied. This finding is consistent with previous reports that the pressure-induced dissociation of erythrocruorin is concentration independent (43), as is the dissociation at 3.5 kbar of myoglobin aggregates initially formed by unfolding at 12 kbar (44). Previous refolding studies at atmospheric conditions report strong negative dependence of recovery of native protein on protein concentration, even when concentrations were very low (e.g., usually 1-50 µg/ml) (14, 33). For example, a study on lysozyme recovery from inclusion bodies required protein concentrations of less than 40 µg/ml (40), while another study on refolding from soluble, denatured lysozyme reported that recovery of active dropped from ca. 95% at 50 µg/ml to ca. 25% at 1 mg/ml (31). Refolding from a denatured (but not aggregated) protein at concentrations above 1 mg/ml has been achieved (45, 46). Only one case has been reported for inclusion body processing at high protein concentrations (47). Methionyl bovine somatotropin inclusion bodies were dissolved and refolded in 4.5 M urea at protein concentrations in the 5-15 mg/ml range with yields of 80% (47).
High pressure has been found to promote formation of molten globule intermediates (48-53). Although it is customary to think of molten globules as highly aggregation-competent conformations, high pressure appears to inhibit this tendency. Work by Smeller et al. (44) has shown that although high pressures (12 kbar) denature myoglobin, aggregates of the unfolded protein do not form until pressure is released. They suggest that intermediate pressures (ca. 3 kbar) populate a folding intermediate that is aggregation prone under atmospheric conditions, but prevented from aggregating by pressure. This suggestion is consistent with the volume change from native state to molten globule intermediate of apomyoglobin of 70 ml/mol (18). Presumably, in the present case, the conformational state(s) of rhGH, lysozyme, and -lactamase that are populated at pressures near 2 kbar all have partial molar volumes that are less than the aggregated state.
