Oxidative Renaturation of Hen Egg-White Lysozyme. Folding vs Aggregation

Abstract

Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species. On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields. Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation. Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation. Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.

High pressure fosters protein refolding from aggregates at high concentrations

“…Then, the correct disulfide bonds must be reformed via air oxidation, an oxido-shuffling system, or exposure to mixed disulfides (16). Most frequently, a mixture of low molecular weight thiols, such as glutathione (oxidized and reduced), is used to reshuffle disulfide bonds during refolding (31). Although these conditions have been shown to be useful in attempts to refold lysozyme (whose native conformation contains four disulfides) from initially soluble, unfolded molecules, studies on lysozyme inclusion bodies have been less successful (40).…”

“…Samples were centrifuged (13,000 g, room temperature) for 15 min to eliminate insoluble aggregates. Extinction coefficients for denatured and native lysozyme are 2.37 and 2.63 (cm mg͞ml) Ϫ1 , respectively (31). Contributions caused by soluble aggregates were subtracted from the signal as reported (30).…”

Pressure treatment of tailspike aggregates rapidly produces on‐pathway folding intermediates

“…For instance, the folding and aggregation of monomeric hen egg-white lysozyme has been modeled as a competition between a first-order productive folding reaction and a third-order aggregation reaction, both from a monomeric intermediate (Maachupalli-Reddy et al, 1997;De Bernardez Clark et al, 1998). This model captures the bulk reaction rates and allowed the researchers to determine the effect of various additives and contaminants on both the folding and aggregation pathways, but stops short of identifying which aggregation intermediates and association steps are most sensitive to the additives.…”

On-Column Refolding and Purification of Recombinant Human Interleukin-1 Receptor Antagonist (rHuIL-1ra) Expressed as Inclusion Body in Escherichia coli

“…Moreover, large volume of liquid would cause much trouble for subsequent operation. Therefore, correct and efficient refolding of inclusion body has been a topic for both basic research and application (De Bernardez Clark et al 1998). Recently, on-column refolding has been developed as an important technique for inclusion body purification (Matsumoto et al 2003;Razeghifard 2004), as solid resin or the folding matrix provided good microenvironment and enough space to prevent improper interactions of polypeptide chains that may lead to aggregation.…”