High pressure fosters protein refolding from aggregates at high concentrations

Abstract

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 mg͞ml) 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 mg͞ml. Inclusion bodies containing ␤-lactamase could be refolded at high yields of active protein, even without added GdmHCl.

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

“…HHP already of pressures as low as 3.5 kbar is sufficient to weaken and (at least partially) disrupt the hydrophobic cores thus leading to formation of a heterogeneous population of fibrillar aggregates with IR amide I' bands in the low wave number region (which is typical of a more strongly H-bonding pattern of intermolecular β-sheets) and a large amount of non-fibrillar smaller aggregates and oligomers, as detected by AFM (with IR amide I' bands in the larger wave number region around 1620 cm -1 ). ii) Our data also indicate that the preformed IAPP fibrils are sensitive to high hydrostatic pressure, similar to amorphous aggregates and inclusion bodies [45][46][47]. Considering the fact that high hydrostatic pressure is an effective means in disturbing ionic and hydrophobic interactions but not hydrogen bonds, we can conclude that these former two types of interaction are important for the stability of IAPP fibrillar aggregates, as also suggested in work using denaturing agents [13].…”

High Hydrostatic Pressure Can Probe the Effects of Functionally Related Ligands on the Quaternary Structures of the Chaperonins GroEL and GroES

“…The application of higher hydrostatic pressure can cause many single chain proteins to denature. A combination of moderate pressure and low concentrations of chaotropes has been found to be suitable for studying the unfolding of proteins (37,38) and for the recovery of proteins from aggregates (39). In addition to providing information on the nature of physical forces involved in the dissociation of oligomeric chaperonins, elucidation of the dissociation mechanism would provide insights into whether such structures would withstand high pressure in bacteria under the depths of the ocean and still be functional for assisting protein folding.…”

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

“…The literature suggests that compression of aggregated proteins at high pressure (2-3 kbar) induces dissociation and refolding [10,26]. However, there are also articles in the literature that indicate that protein folding occurs during incubation at lower pressure levels (0.3-0.7 kbar) [13,27] or at atmospheric pressure [9].…”

“…Application of 1-3 kbar disrupts the http://dx.doi.org/10.1016/j.pep.2014. 10.013 1046-5928/Ó 2014 Elsevier Inc. All rights reserved. intermolecular electrostatic and hydrophobic interactions, dissociating oligomeric and aggregated proteins.…”

“…However, more efficient protein hydration followed by disruption of intramolecular bonds and subsequent protein denaturation generally requires pressure higher than 4 and 5 kbar [8]. Therefore, the use of 2-3 kbar constitutes a non-denaturing technique that solubilizes aggregates and keeps the secondary and tertiary protein structure intact, being potentially applicable in protein refolding [9,10]. In this context, HHP has become a technique to obtain high-yield protein refolding [11][12][13][14].…”