The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to sl...

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in...

The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final refolded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (DK-SS) were used to dissolve denatured HEWL without denaturants such as guanidine hydrochloride (GuHCl) and urea. When denatured HEWL was perfectly dissolved in buffer solutions con...

In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli. This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results.

The impact of the quenched force on protein folding pathways and free energy landscape was studied in detail. Using the coarse-grain Go model, we have obtained the low, middle, and high force regimes for protein refolding under the quenched force. The folding pathways in the low force regime coincide with the thermal ones. A clear switch from thermal folding pathways to force-driven pathways in...

Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have been used for antibody fragments, comprising dilution, dialysis, sol...

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchro...

Refolding of proteins at high concentrations often results in aggregation. To gain insight into the molecular aspects of refolding and to improve the yield of active protein, we have studied the refolding of lysozyme either from its denatured state or from its denatured/reduced state. Refolding of denatured lysozyme, even at 1 mg/ml, yields fully active enzyme without aggregation. However, refo...

Three foldases--the apical domain of GroEL (mini-chaperone) and two oxidoreductases (DsbA and DsbC) from Escherichia coli--were studied in refolding a protein with immunoglobulin fold (immunoglobulin-folded protein) that had been produced as inclusion bodies in E.coli. The foldases promoted the refolding of single-chain antibody fragments from denaturant-solubilized and reduced inclusion bodies...

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of variou...