• Journal of Microbiology and Biotechnology
• /
• v.6 no.6
• /
• pp.456-460
• /
• 1996

Recombinant Pseudomonas lipase was used to study protein aggregation and adsorption upon in vitro refolding. Protein adsorption as well as aggregation was responsible for major side reactions upon in vitro refolding as a function of protein concentration. The optimal range of protein concentration was determined by the relative contribution of protein aggregation and adsorption. Above the optimal range, the yield of active lipase inversely correlated with protein aggregation, showing a competition between folding and aggregation. However, adsorption of protein rather than protein aggregation is thought to contribute as a major side reaction of the refolding process at sub-optimal concentrations at which the formation of aggregates should be more reduced. Protein aggregation was influenced by the amount of guanidine hydrochloride in the refolding solvent. The refolding temperature was a critical factor determining the extent of protein aggregation. The refolding yield was also affected by the dilution fold and dilution mode, which suggests that the refolding process might kinetically compete with the rate of mixing.

• KSBB Journal
• /
• v.16 no.1
• /
• pp.11-14
• /
• 2001

Techniques for protein refolding from inclusion body are discussed in view of its engineering application to large scale protein purification. Among the techniques, dilution and dialysis are mainly utilized due to simple operation. Membrane reactor, gel filtration chromatography, and continuous tank operation are emerging tools for their process-scale possibility in refolding. Reaction engineering approaches could be used to analyze the kinetic behaviour in the process scale refolding reactor. The kinetic analysis is helpful in the optimization of refolding yield in the refolding reactor.

• Journal of the Korean Magnetic Resonance Society
• /
• v.23 no.4
• /
• pp.104-107
• /
• 2019

Many proteins are expressed as an insoluble form during the production using Escherichia coli (E. coli) system. Although various methods are applied to increase their amounts of soluble expression, refolding is the only feasible way to obtain a target protein in some cases. Moreover, protein NMR experiments require ¹³C/¹⁵N-labeled proteins that can only be obtained from E. coli systems in terms of cost and technical difficulty. The finding of appropriate refolding conditions for a target protein is a time-consuming process. In particular, it is very difficult to determine whether the refolded protein has a native structure, when a target protein has no enzymatic activity and its refolding yield is very low. Here, we showed that 1-dimensional ¹H-¹⁵N heteronuclear single quantum correlation (1D ¹H-¹⁵N HSQC) experiment can be efficiently used to screen an optimal condition for the refolding of a target protein by monitoring both the structure and concentration of the refolded protein.

• KSBB Journal
• /
• v.13 no.2
• /
• pp.133-140
• /
• 1998

Using rlFN-$\alpha$ and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

• Proceedings of the Korean Society of Life Science Conference
• /
• 2002.09a
• /
• pp.9-17
• /
• 2002

Renaturation of Lysozyme by size exclusion chromatography(SEC) to improve yield as well as the initial and final protein concentration has been studied in detail, Although urea decreases the rate of proteins refolding, it can suppress protein aggregation to sustain pathway of correct refolding at high protein concentration, and there existed an optimum urea concentration in renaturation buffer. Lysozyme was successfully refolded from initial protein concentration of up to 100mg/m1 by SEC, the yield was more than 40%. And the refolding of Interferon-${\gamma}$ was further investigated.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.10
• /
• pp.1742-1750
• /
• 2015

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at $15^{\circ}C$, whereas the reaction at $25^{\circ}C$ was faster than that at $15^{\circ}C$. The refolding yield at $15^{\circ}C$ was 17% higher than that at $25^{\circ}C$. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at $15^{\circ}C$ for 16 h. The maximum refolding yield was 74.8% at $15^{\circ}C$ with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.7 no.1
• /
• pp.1-5
• /
• 2002

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.6
• /
• pp.500-504
• /
• 2005

Fusion ferritin (heavy chain ferritin, $F_H+$ light chain ferritin, $F_L$), an iron-binding protein, was primarily purified from recombinant Escherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, and L-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclusion bodies (IBs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.

• KSBB Journal
• /
• v.16 no.2
• /
• pp.146-152
• /
• 2001

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we propose a solid-phase refolding method integrated with expanded bed adsorption chromatography. The model protein used was a fusion protein of recombinant human growth hormone and a glutathione S transferase fragment. It was demonstrated that the EBA-mediated refolding technique could simultaneously remove cellular debris and directly renature the fusion protein inclusion bodies in the cell homogenate with much higher yields and less agregation. To demonstrate the applicability of the method, we successfully tested the three representative types of starting materials, i. e., rhGH monomer, washed inclusion bodies, and the E. coli homogenate. This direct and simplified refolding process could also reduce the number of renaturation steps required and allow refolding at a higher concentration, at approximately 2 mg fusion protein per ml of resin. To the best of our knowledge, it is the first approach that has combined the solid-phase refolding method with expanded bed chromatography.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.6
• /
• pp.410-418
• /
• 2001

The effects of several variables on the refolding of hen egg white lysozyme have been studied, Lysozyme was denatured in both urea, and guanidine hydrochloride(GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1 M Tris-HCI, pH 8.2 mM EDTA 3 mM reduced glutathione and 0.3 mM oxidised glutathions. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50$\^{C}$. The apparent activation energy for lysozyme re-folding wasf ound to be 56kJ/mol, Refolding by dilution results in low concentrations of both de-naturant and reducing agent species. It was found that the residual concentrations obtained dur-ing dilution(100-fold dilution:[GuHCI]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1-10mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT higher refolding yields were obtained when starting from higher initial lysozyme concentra-tions. This trend was reversed when residual denaturant components were removed from the re-folding buffer.