Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

包涵体蛋白的分离和色谱法体外复性纯化研究进展

重组蛋白在大肠杆菌中表达多为无活性的包涵体形式,须经洗涤、溶解、复性后才能得到生物活性蛋白。综述了近年来包涵体蛋白分离纯化和复性技术研究进展,重点讨论了色谱法复性技术的应用,包括尺寸排阻色谱、亲和色谱、离子交换色谱、疏水相互作用色谱、固定化脂质体色谱、扩张床吸附色谱的进展情况。     

High productivity refolding of an inclusion body protein using pulsed-fed size exclusion chromatography

Protein refolding using size exclusion chromatography (SEC) is advantageous over conventional refolding methods in terms of ease of automation, simultaneous purification capabilities, and the non-adsorptive protein–matrix interaction which eliminates steric constraints. Despite these advantages, the widespread use of SEC refolding remains restricted by low process productivity and product concentration bottlenecks. This study aims to address those limitations and exploit SEC advantages for large-scale refolding applications. Specifically, this study reports the development of a pulsed-fed size exclusion chromatography (PF-SEC) refolding platform, which successfully refolded E. coli-derived α-fetoprotein (AFP) to achieve 53% refolding yield at 0.9 mg/ml AFP refolding concentration. AFP was introduced into the column by pulsed injection to increase feed load, while suppressing concentration-induced aggregation. Refolding was initiated by a urea gradient in the column, where the gradient length could be readily adjusted to complement pulsed feeding patterns. AFP refolding productivity with PF-SEC improved by 8- and 64-fold compared to ion-exchange chromatography refolding and pulsed dilution refolding, respectively, at a fixed refolding concentration. Through a unique integration of pulsed feeding and urea gradient development, this new PF-SEC refolding methodology overcomes ‘productivity and concentration’-related disadvantages inherent in SEC, and will be scalable for large-scale protein refolding applications.     

蛋白质的排阻色谱复性的新进展

外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。     

Improving the refolding of NTA protein by urea gradient and arginine gradient size-exclusion chromatography

Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme.     

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.     

Refolding of pyridoxine-5'-P oxidase assisted by GroEL

The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.     

Refolding of recombinant human interferon ��-2a from Escherichia coli by urea gradient size exclusion chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon ??-2a (rhIFN??-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhIFN??-2a would pass along the 8.0?C3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 × 10⁸ IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhIFN??-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhIFN??-2a refolding in terms of specific activity and mass recovery.     

Effect of column dimensions and flow rates on size-exclusion refolding of beta-lactamase

We have investigated the effect of changing the column diameter and length on the size exclusion chromatography (SEC) refolding of beta-lactamase from Escherichia coli-derived inclusion bodies (IBs). Inclusion bodies were recovered and solubilised in 6 M GdnHCl and 5 mM DTT. Up to 16 mg of denatured, solubilised beta-lactamase was loaded onto size exclusion columns packed with Sephacryl S-300 media (fractionation range: 10(4)-1.5 x 10(6) Da). beta-Lactamase was refolded by eluting the loaded sample with 1 M urea in 0.05 M phosphate buffer, pH 7 at 23 degrees C. The following columns were studied: 26 x 400, 16 x 400 and 26 x 200 mm, with a range of mobile phase flow rates from 0.33 to 4.00 ml/min. beta-Lactamase was successfully refolded in all three columns and at all flow rates studied. The beta-lactamase activity peak coincided with the major protein peak. Reducing the column diameter had little effect on refolding performance. The enzyme activity recovered was relatively independent of the mobile phase linear velocity. Reducing the column length gave a poorer resolution of the protein peaks, but the enzyme activity peaks were well resolved. Calculation of the partition coefficients for beta-lactamase activity showed that the 26 x 400 column gave the greatest refolding performance.     

Folding pathway for partially folded rabbit muscle creatine kinase.

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.     

Protein refolding at high concentration using size-exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. (c) 1996 John Wiley & Sons, Inc.     

层析辅助体外复性含有多对二硫键的融合蛋白质

为建立一种基于阴离子交换介质辅助的含多对二硫键的抗凝溶栓双功能水蛭素12肽-瑞替普酶融合蛋白质 (HV12p-rPA) 的复性方法,采用Q Sepharose XL作为层析复性介质,通过正交实验考察蛋白质上样量、流速、脲梯度、洗脱液中精氨酸浓度、脲浓度、pH、还原型及氧化型谷胱甘肽等因素对复性过程的影响,探索最佳层析复性条件。结果表明：脲梯度、上样量及精氨酸浓度是影响复性的3个主要因素。脲梯度是复性成功的关键,上样量增大时复性蛋白质比活降低,精氨酸辅助HV12p-rPA复性的最佳浓度为1 mol/L。创建了脲、pH双梯度下的阴离子交换层析辅助HV12p-rPA的复性方法,复性后蛋白质的溶栓比活达到46 520 IU/mg,抗凝比活达到9 980 ATU/mg,与稀释复性方法相比,该方法能使复性蛋白质的溶栓比活提高14~15倍,抗凝比活提高7~8倍。     

The effect of polyols on the reactivation of guanidium chloride-denatured arginine kinase from shrimp feneropenaeus chinensis muscle

The influence of glycerol, sorbitol, glucose and sucrose, on the refolding and reactivation courses of guanidine-denatured arginine kinase was studied. Glycerol, sucrose, and sorbitol, but not glucose, could improve the reactivation of the denatured arginine kinase, although in all cases aggregation was inhibited. Size exclusion chromatography showed that misfolded products were still formed during polyol-assisted refolding. The chemical and physical characteristics of polyols might explain the various observations.     

High recovery refolding of rhG-CSF from Escherichia coli, using urea gradient size exclusion chromatography

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.     

The role of detergent in refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus Chinensis: the solubilization of aggregate and refolding in detergent solutions.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.     

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.     

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Chaperon solvent plug design in size-exclusion chromatography protein refolding process

Although the chaperon solvent plug was reported as a strategy to reduce aggregation before the column inlet in SEC (size-exclusion chromatography) protein refolding process, the appropriate position at which sample injected and the volume of the chaperon solvent plug have not been elucidated. Therefore, the detail of chaperon solvent plug design was investigated in this work. Our results indicated that, to ensure good performances in the SEC refolding process, the appropriate front and tail volumes of chaperon solvent plug should be slightly larger than the optimal values, which depend on the flow dispersion from the injector to the column inlet. However, with the front volume more than the optimum, it could have an adverse effect on activity recovery but not the mass recovery, while no effect at all if the tail volume exceeded the optimum. Furthermore, it might be economical to replace the eluent (refolding buffer) after the tail of chaperon solvent plug with a cheaper one.     

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.     

A novel "reverse screening" to identify refolding additives for activin-A

A general approach for refolding recombinant proteins from inclusion bodies (IBs) is to screen conditions, that facilitate a conversion of unfolded to folded structure and minimize a conversion of unfolded to misfolded and aggregated structures. In this simplified model, such conditions may be those that stabilize the native protein and/or reduce aggregation. In this paper, a novel screening approach, termed reverse screening, was developed using a native activin. Activin-A, a member of transforming growth factor beta superfamily, is a homodimeric protein with nine disulfide bonds. We examined partial unfolding process of native activin-A dissolved in a buffer containing moderate concentrations of denaturant and reducing reagent (i.e., 1.5 M urea and 0.2 mM dithiothreitol). The recovery of the protein was followed by reverse-phase high performance chromatography analysis. Without additives, activin-A showed about 60% loss of the protein due to aggregation after 12-h incubation in the above condition. We then tested various additives for their effects on the recovery after partial unfolding. One of these additives, sodium taurodeoxycholate (TDCA), greatly increased recovery and suppressed aggregation of the protein. These additives were then tested for refolding activin-A from IBs. TDCA among others is proved to be a highly effective refolding additive. These results strongly suggest that reverse screening using native proteins, if available, may be another approach to discovering effective refolding additives.