Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using ＂Phase Diagram＂ Method of Fluorescence

The refolding of reduced and non-reducing egg white lysozymes in a urea solution was studied by a ＂phase diagram＂ method of fluorescence. The result showed that in the refolding of the reduced egg white lysozyme, an intermediate state of an egg white lysozyme exists at the urea concentrations in a final renaturation solution being about 4.5 mol/L, their refolding follows a three-state model; while in the refolding of the non-reducing egg white lysozyme, two intermediate states exist at the urea concentrations being separately 4.0 and 2.5 mol/L, and their refolding follows a four-state model. Through the comparison between the unfolding and refolding of an egg white lysozyme in the urea solution, it was found that both of the refolding of reduced and non-reducing egg white lysozyme molecules was irreversible to their unfolding in the urea solution. Finally, a suggested refolding was separately presented for the reduced and non-reducing egg white lysozymes in the urea solution.     

pH-sensitive polymer-assisted refolding of urea-denatured fibroblast growth factor

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of FGF-2(fibroblast growth factor-2) denatured with 8 mol/L urea and 10 mmol/L dithiothreitol at pH 7.2.The refolding of FGF-2 was performed by directly diluting denatured FGF-2 into a refolding buffer containing Eudragit S-100.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTr method,fluorescence emission spectroscopy and reverse phase HPLC.On the other hand,the result shows the ab...     

溶液中变性剂浓度对蛋白溶菌酶复性的影响

Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n（m1 -m2） and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography.     

Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography(WAX) coupled with MALDI-TOF MS.The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX.However,with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1:6 in the mobile phase employed,the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the ...     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Enhanced refolding of lysozyme with imidazolium-based room temperature ionic liquids: Effect of hydrophobicity and sulfur residue

The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies (IBs). Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids (RTILs) on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4]-based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to the conventional refolding buffer, 2 times higher lysozyme refolding yield was obtained in 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) containing refolding buffer. The refolding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MS]) containing sulfur residue on anion was used. The sulfur residue in [BMIM][MS] is supposed to improve the refolding yield of lysozyme which has 4 intramolecular disulfide bonds. For dilution-based refolding of lysozyme, the optimum concentrations of RTILs in refolding buffer were found to be 1.0 M [EMIM][BF4] and 0.5 M [BMIM][MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 °C.     

Time-resolved ESI-MS Investigation on Reduction and Alkylation in Unfolding Process of Lysozyme

The kinetic procedure of the unfolding of lysozyme induced by the reduction of disulfide was monitored by the time-resolved ESI-MS with a sheath liquid assistant electrospray interface. It was found that the reduction process for the eight disulfides had a less difference in the reaction time after denatured treatment. In addition, the alkylation of the reduced free thiols was much slower than the reduction procedure. An artifact peak produced by the CID fragmentation in the mass spectra was identified and the possible mechanism of the Hofmann elimination reaction was proposed.     

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCI) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disul-fide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with re-versed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.     

Preparation of Medium Cation Exchange Stationary Phase of Polymeric Matrix and Their Chromatographic Properties

Based on the monodisperse poly（glycidyl methacrylate-co-ethylenedimethacrylate） beads （PGMA/EDMA） with macropore as a medium, a new hydrophilic medium cation exchange （MCX） stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic （IEC） retention mechanism. The measured bioactivity recovery for lysozyme was （96 ± 5）%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.     

Refolding of detergent‐denatured lysozyme using β‐cyclodextrin‐assisted ion exchange chromatography

Chromatography‐based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent‐denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl‐denatured/dithiothreitol‐reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β‐cyclodextrin, was developed for refolding detergent‐denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β‐cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl‐denatured lysozymes. The results showed that the sarkosyl‐denatured lysozyme could be successfully refolded using β‐cyclodextrin‐assisted ion exchange chromatography. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of zeolite to refold a disulfide-bonded protein

Zeolites are microporous crystalline aluminosilicates with a highly ordered structure. Using zeolite beta as an adsorbent, denatured/reduced hen egg lysozyme was refolded to the active form at high concentrations. The denatured/reduced lysozyme was adsorbed onto the zeolite and the protein was refolded by desorbing it into refolding buffer, consisting of redox reagents, guanidine hydrochloride, polyethylene glycol, and L-arginine. This zeolite refolding method could be highly effective for various kinds of proteins, refolding them with high efficiency even when they contain disulfide bonds.     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

Protein refolding assisted by periodic mesoporous organosilicas

Herein we report a new strategy for protein refolding by taking advantage of the unique surface and pore characteristics of ethylene-bridged periodic mesoporous organosilica (PMO), which can effectively entrap unfolded proteins and assist refolding by controlled release into the refolding buffer. Hen egg white lysozyme was used as a model protein to demonstrate the new method of protein refolding. Through loading of denatured proteins inside uniform mesoporous channels tailored to accommodate individual protein, protein aggregation was minimized, and the folding rate was increased. Poly(ethyleneglycol) (PEG)-triggered continuous release of entrapped denatured lysozyme allowed high-yield refolding with high cumulative protein concentrations. The new method enhances the oxidative refolding of lysozyme (e.g., over 80% refolding yield at about 0.6 mg/mL).     

温敏型聚合物PNIPAAm辅助的溶菌酶体外复性

合成了 3种具有不同分子量的温敏型聚合物聚 (N 异丙基丙烯酰胺 ) (PNIPAAm) ,测定了其分子量分布以及相应的低临界溶解温度 (LCST) .在溶菌酶复性溶液中加入PNIPAAm可促进溶菌酶复性 ,其中采用中等分子量M—PNIPAAm(Mw 为 2 2× 10 4 g mol)时溶菌酶的复性效果最佳 ,并采用荧光发射光谱技术表征了PMIPAAm分子结构对于溶菌酶结构的影响 .系统考察了采用M—PNIPAAm时 ,复性液中尿素浓度、蛋白质浓度和温度等条件对溶菌酶复性效果影响 .结果显示尿素与M—PNIPAAm对于溶菌酶复性呈现协同效应 ,复性操作温度不仅同溶菌酶自身特性有关 ,而且还受到M—PNIPAAm自身性质变化的影响 .研究结果表明温敏型高聚物在高浓度蛋白质的大规模体外复性中具有很好的应用前景     

Considerations of sample application and elution during size-exclusion chromatography-based protein refolding

A mechanism for size-exclusion chromatography-based protein refolding is described. The model considers the steps of loading the denatured protein onto a gel filtration column, and protein elution. The model predictions are compared with results of refolding lysozyme (10 and 20 mg/ml) using Superdex 75 HR. The main collapse in protein structure occurred immediately after loading, where the partition coefficient of unfolded lysozyme increased from 0.1 to 0.48 for the partially folded molecule. Use of a refolding buffer as the mobile phase resulted in complete refolding of lysozyme; this eluted at an elution volume of 15.6 ml with a final partition coefficient of 0.54. The model predicted the elution volume of refolded lysozyme at 19.3 ml.     

Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles

The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.     

Synthesis of Monodisperse Poly（glycidylmethacrylate-co-ethylene dimethacrylate） Beads and Their Application in Separation of Biopolymers

Introduction Since 19541 the polymeric separation media has attracted much attention due to their chemical stability over the entire pH range. The rigid, highly cross-linked styrene copolymers were first used for chromatography by Moore.2 The macroporous copolymers currently available are not only chemically stable but also more resistant to mechanical forces prevailing in a column and therefore are comparable to the traditional packings based on silica gel. Most polymer separation media are …     

Synthesis of monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads and their application in separation of biopolymers

The monodisperse, porous poly(chloromethylstyrene-co-divinylbenzene) beads of 7.9 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation-exchange (WCX) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery, and good resolution for proteins. The dynamic protein-loading capacity of the synthesized WCX packings was 18.2 mg/g. Five proteins were separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 5.0 min with only one step. The purity and specific bioactivity of the purified lysozyme were found to be more than 93% and 70 245 U/mg, respectively.     

Studies on Aggregation Interaction between Reduced Denatured Egg White Lysozymes during Refolding Procedure in Urea Solution

The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.