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.     

One‐step refolding and purification of recombinant human tumor necrosis factor‐α (rhTNF‐α) using ion‐exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic‐based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor‐α (rhTNF‐α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF‐α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF‐α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10⁸ IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF‐α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF‐α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.     

On‐column refolding of corticotropin‐releasing factor receptor 1 extracellular domain by size exclusion chromatography

Expressing the extracellular domain of corticotropin releasing factor receptor 1 in Escherichia coli usually results in the formation of inclusion bodies. Here we describe the optimization of refolding by applying size exclusion chromatography with a denaturing guanidine hydrochloride gradient and a refolding buffer containing glycerol. Several chromatographic parameters like gradient length, flow rate, sample concentration and chromatography resin characteristics were evaluated. Recovery yields of refolded protein above 50% using a Superdex 200 column demonstrate the usefulness of this method. Copyright © 2009 John Wiley & Sons, Ltd.     

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Protein‐folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high‐performance size‐exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low‐urea gradient‐elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS–18% PAGE, Matrix‐Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI‐TOF‐MS) and the biological activity assay by HP‐RPLC. Copyright © 2014 John Wiley & Sons, Ltd.     

Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

谷胱甘肽键合柱对变性核糖核酸酶的复性研究

将氧化还原型谷胱甘肽(GSH/GSSG)共价键合到色谱固定相上, 实现了对变性核糖核酸酶(RNase)的复性. 实验发现, 谷胱甘肽键合柱具有典型的弱阳离子交换性质, 在离子交换(IEC)模式下能够对4种标准蛋白进行基线分离, 且具有较高的柱效. 当蛋白浓度为5 mg/mL, 流速为0.2 mL/min时, 在流动相中不加GSH/GSSG的条件下, GSH/GSSG柱对变性核糖核酸酶的活性回收率可达(39.5±3.8)%, 而普通IEC柱对变性核糖核酸酶的活性回收率几乎为0, 说明其对变性蛋白二硫键的正确对接具有明显的促进作用; 在收集液中加入GSH/GSSG后, 其活性回收率可达到(81.5±4.3)%. 本文结果对蛋白折叠液相色谱法的发展及降低蛋白复性成本具有一定的应用价值.     

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.     

On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption

Protein refolding at high concentrations always leads to aggregation, which limits commercial application. An ion-exchange chromatography process with gradient changes in urea concentration and pH was developed to refold denatured lysozyme at high concentration. After adsorption of the denatured protein onto an ion-exchange medium, elution was carried out in combination with a gentle decrease in urea concentration and elevation of pH. Protein would gradually refold along the column with high activity yield. Denatured and reduced lysozyme at 40 mg/ml was loaded into a column filled with SP Sepharose Fast Flow, resulting in 95% activity recovery and 98% mass yield within a short period of time.     

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.     

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

In this work, based on the structural characteristics of bio‐membrane molecules, a novel type of high‐performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one‐step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.     

Refolding of urea‐denatured α‐chymotrypsin by protein‐folding liquid chromatography

An approach for re‐folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α‐chymotrypsin (α‐Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α‐Chy states – urea‐denatured (U state), its folded intermediates (M state) and nature state (N state) – were studied during protein folding. Based on the test by matrix‐assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α‐Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α‐Chy was found to be higher than that of commercial α‐Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m ; the highest specific bioactivity at urea concentration was 1.0 m , indicating the possibility for re‐folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α‐Chy. When the urea concentration reached 6.0 m , the unfolded α‐Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.     

Novel column-based protein refolding strategy using dye-ligand affinity chromatography based on macroporous biomaterial

A novel column-based chromatographic protein refolding strategy was developed using dye-ligand affinity chromatography (DLAC) based on macroporous biomaterial. Chitosan–silica (CS–silica) biomaterial with macroporous surface was used as the supporting matrix for the preparation of the DLAC material. The dye-ligand Cibacron Blue F3GA (CBF) was selected as affinity handle and could be covalently immobilized to form dye-ligand affinity adsorbent (CBF–CS–silica) using the reactivity of NH₂ on CS–silica biomaterial. After the model protein catalase was denatured with 6 mol/L urea, the denaturant could be rapidly removed and catalase could be successfully refolded as facilitated by the adsorption of CBF–CS–silica. The urea denaturation process and the elute condition for the chromatographic refolding were optimized by measuring tryptophan fluorescence and activity of catalase. The refolding performance of the proposed DLAC was compared with dilution refolding. The protein concentration during the proposed chromatographic refolding increased by a factor of 20 without reducing the yield achieved as compared to dilution refolding. The column-based protein refolding strategy based on dye-ligand affinity chromatography with porous biomaterial being matrix possessed potential in chromatographic refolding of protein.     

体积排阻色谱法研究变性溶菌酶分子复性过程中集聚体的形成

在体积排阻色谱柱上研究了还原剂存在时脲和盐酸胍变性的3种溶菌酶溶液的复性和分离过程。当变性液中原始溶菌酶浓度大于10 g/L时,变性溶菌酶在体积排阻色谱柱上除了复性为与未变性溶菌酶出峰时间相同的复性态溶菌酶分子外,还形成了溶菌酶折叠中间体的二分子集聚体。这个结果得到了用稀释法复性时溶菌酶的蛋白电泳检测结果的支持。与稀释法复性相比较,用体积排阻色谱法复性时所形成的折叠中间体二分子集聚体的量要远远低于用稀释法所形成的集聚体的量。     

Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.     

Lysozyme refolding with immobilized GroEL column chromatography

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.     

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^t-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 disulfide 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 reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

蛋白折叠液相色谱法复性和纯化大肠杆菌表达的重组人粒细胞集落刺激因子

用蛋白折叠液相色谱法(PFLC)对大肠杆菌表达的包涵体形式的重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化。用Cu2+-亚氨基二乙酸（IDA） Sepharose作为固定化金属离子亲和色谱的固定相。在低浓度脲存在下,以咪唑为洗脱剂,采用线性梯度洗脱rhG-CSF。该法仅通过一步PFLC分离,减少了复性过程中rhG-CSF的聚集,复性后的rhG-CSF的比活性为1.8×108 IU/mg,纯度为97%,质量回收率为32%。     

采用不同疏水相互作用色谱柱从包涵体中快速制备重组人干细胞因子

为了提高重组人干细胞因子(rhSCF)的复性效率,改进了高效疏水相互作用色谱(HPHIC)纯化和复性rhSCF的方法。首先将目标蛋白溶解于8.0 mol/L脲中,然后将rhSCF包涵体的提取液直接进样到不同规格的HPHIC柱进行纯化和复性。优化了固定相配基结构和流动相组成等实验条件,结果表明,本方法可以快速地获得高质量回收率和高生物活性的rhSCF,rhSCF在40 min内即可完成复性与纯化,目标蛋白的纯度在95.5%以上,质量回收率高于49.6%。通过体积排阻色谱和基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)的分析,确认rhSCF以单体存在。结果进一步证明HPHIC法是同时复性和纯化重组蛋白的有效工具。