In general, protein aggregation is considered an irreversible process. A large variety of techniques have been examined in industry to refold proteins from aggregates and inclusion bodies. Discovery of a successful refolding method usually requires extensive investigation of a host of chemical additives during removal of chaotrope such as GdmHCl or 10 M urea. Often, refolding yields are low despite extensive effort.; Pressure is known to unfold proteins. In general, multimeric proteins unfold between 1–3 kbar, and monomers unfold between 4–8 kbar. Assuming that an aggregate is essentially a large, multimeric protein complex, we expect to observe a pressure window in which the protein aggregate complex is unstable, while native monomers remain stable. Hence, pressure will effectively refold proteins from aggregates.; In our initial studies, we found that high hydrostatic pressures (1–2 kbar) combined with low, nondenaturing concentrations of guanidine hydrochloride (GdnHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone (rhGH) and lysozyme, and β-lactamase inclusion bodies. 100% 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 0.75M guanidine hydrochloride. 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 β-lactamase could be refolded at high yields of active protein, even without added guanidine hydrochloride.; With covalently-crosslinked aggregates of lysozyme, non-denaturing levels of guanidine hydrochloride (GdnHCl) improves refolding yields to 80% at 1M GdnHCl; higher concentrations do not increase refolding yields further. Varying glutathione redox ratios results in a maximum refolding yield near 1:1 oxidized to reduced glutathione (GSSG:GSH). Yields drop off dramatically at more oxidizing conditions ([GSSG] > [GSH]), where more reducing environments exhibit a more subtle decrease in refolding yields. Kinetics of refolding covalently crosslinked aggregates of lysozyme depend strongly on redox conditions. Structural analysis reveals near complete destruction of native secondary structure in the aggregate. Hence, aggregated lysozyme transitions through a large structural shift to reform native secondary structure and native disulfide bonds while under pressure. Once refolded while under pressure, reaggregation upon depressurization is highly unlikely. (Abstract shortened by UMI.)
展开▼
