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.     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 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. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for 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 HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

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.     

On-column refolding of consensus interferon at high concentration with guanidine-hydrochloride and polyethylene glycol gradients

Dilution refolding of consensus interferon (C-IFN) had a limit on final concentration not exceeding 0.1 mg ml(-1) in order to achieve specific activity of 2.2x10(8) U mg(-1). Addition of polyethylene glycol (PEG) only gave a marginal improvement on the specific activity. Hydrophobic interaction chromatography (HIC) was tried but a simple step-wise elution could not refold the protein. Successful refolding was achieved by gradient elution with the decreasing of guanidine-hydrochloride (guanidine-HCl) concentration. The column was packed with a commercially available HIC medium that was designed for protein separation. Polyethylene glycol was found to possess better effect on the column than in the dilution for promotion of correct refolding, especially in gradient mode. A novel dual-gradient strategy, consisting of decreasing guanidine-HCl concentration and increasing PEG concentration, was developed to enhance the refolding yield. Denatured C-IFN was allowed to adsorb and elute from the HIC column through a gradually changed solution environment. Compared with dilution refolding, the gradient HIC process, in the presence of PEG, gave about 2.6-folds of increase in specific activity, 30% increase in soluble protein recovery. Partial purification was also achieved simultaneously.     

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.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

Continuous chromatographic protein refolding

Column-based protein refolding requires a continuous processing capability if reasonable quantities of protein are to be produced. A popular column-based method, size-exclusion chromatography (SEC) refolding, employs size-exclusion matrices to separate unfolded protein from denaturant, thus refolding the protein. In this work, we conduct a comparison of SEC refolding with refolding by batch dilution, using lysozyme as a model protein. Lysozyme refolding yield was found to be extremely sensitive to the chemical composition of the refolding buffer and particularly the concentration of dithiothreitol (DTT) introduced from the denatured protein mixture. SEC refolding was not adversely affected by DTT carry-over as small contaminants in the denatured solution are separated from protein during the refolding operation. We also find that, contrary to previous reports, size-exclusion refolding on batch columns leads to refolding yields slightly better than batch dilution refolding yields at low protein concentrations but this advantage disappears at higher protein concentrations. As batch-mode chromatography would be the limiting step in a column based refolding downstream process, the batch column refolding method was translated to a continuously operating chromatography system (preparative continuous annular chromatography, P-CAC). It was shown that the P-CAC elution profile is similar to that of a stationary column, making scale-up and translation to P-CAC relatively simple. Moreover, it was shown that high refolding yields (72%) at high protein concentration (>1 mg ml(-1)) could be obtained.     

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N^pro autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO₃⁻, UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris–HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO₃⁻, refolding could be performed at column loadings of 50 mg fusion protein/ml gel, resulting in a final eluate concentration of around 10–15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.     

蛋白折叠液相色谱法中流动相对重组人干扰素-γ质量回收率的影响

蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4(pH 7.0)和0.05 mol/L KH2PO4(pH 7.0)为流动相A和B,采用35 min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。     

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.     

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).     

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.     

Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

重组人干扰素-γ的制备与鉴定

用聚乙二醇200疏水相互作用色谱固定相(PEG200-STHIC)分别在色谱柱和色谱饼上完成了一步复性并同时纯化来源于大肠杆菌(E.coli)表达的重组人干扰素-γ(rhIFN-γ)。为了能使色谱分离方法用于不同来源的rhIFN-γ的纯化,对rhIFN-γ在反相色谱、离子交换色谱、固定化镍离子亲和色谱上的保留行为也进行了研究。色谱柱纯化的rhIFN-γ收集液经排阻色谱除盐和冷冻干燥得到rhIFN-γ干粉。用基质辅助激光解吸电离飞行时间质谱对rhIFN-γ干粉进行了测定,rhIFN-γ单体的相对分子质量为17184.0,二聚体的相对分子质量为34204.4。用细胞病变抑制法(CPEI)测定rhIFN-γ干粉的比活性为9.5×108 IU/mg。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定rhIFN-γ干粉的纯度高于95%。用色谱柱复性并同时纯化rhIFN-γ的质量回收率达到93.7%,纯度高于95%,比活性为4.3×107 IU/mg。结果表明,采用PEG200-STHIC色谱柱复性并同时纯化rhIFN-γ是一种十分高效的方法。     

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.     

Characterization of the refolding and reassembly of an integral membrane protein OmpF porin by low-angle laser light scattering photometry coupled with high-performance gel chromatography

The refolding and reassembly of an integral membrane protein OmpF porin denatured in sodium dodecylsulfate (SDS) into its stable species by the addition of n-octyl-beta-D-glucopyranoside (OG) have been studied by means of circular dichroism (CD) spectroscopy and low-angle laser light scattering photometry coupled with high-performance gel chromatography. The minimal concentration where change in the secondary structure was induced by the addition of OG was found to be 6.0 mg/ml in CD experiments. A species unfolded further than the SDS-denatured form of this protein was observed at an early stage (5-15 min) of refolding just above the minimal OG concentration. In addition, the CD spectrum of protein species obtained above the minimal OG concentration showed that the protein is composed of a beta-structure which is different from the native structure of this protein. In light scattering experiments, no changes in molecular assemblies were observed when the OG concentration was below its minimal refolding concentration determined by CD measurements. Above the minimal concentration, a compact monomeric species was observed when denatured OmpF porin was incubated for 5 min at 25 degrees C in a refolding medium containing 1 mg/ml SDS and 7 mg/ml OG, and then injected into columns equilibrated with the refolding medium. After an incubation of 24 h before injection into the columns, predominant dimerization of this protein was observed in addition to incorrect aggregation.     

Production, purification and characterization of recombinant human interferon gamma.

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.     

Measurement of daphnoretin in plasma of freely moving rat by liquid chromatography

Daphnoretin (7-hydroxyl-6-methoxy-3,7'-dicoumaryl ether), isolated from Wikstronemia indica C.A. Mey. (Thymelaceae), has been reported to induce rabbit platelet aggregation through protein kinase C activation and anticancer activity. In this study, we developed an automated blood sampling system coupled to a simple and sensitive HPLC system to determine plasma concentration of daphnoretin in rats. This method was applied to investigate the pharmacokinetics of daphnoretin in a freely moving rat. Separation of daphnoretin in the rat plasma was achieved using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of methanol-10 mM NaH2PO4 (adjusted to pH 3.0 with H3PO4) (55:45, v/v), and the flow rate of 1.0 ml/min. The UV detector was set at 345 nm. The automated blood sampling system (DR-II has been applied for blood sampling in a conscious and freely moving rat. The blood samples were centrifuged at 3000 x g for 10 min and the plasma samples were then deproteinized by acetonitrile containing an internal standard (khellin 1 microg/ml). After centrifugation (8000 x g for 10 min), the aliquot of supernatant was injected into the HPLC system for analysis. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.05-1.00 and 1.00-100 microg/ml. Intra- and inter-assay precision and accuracy of daphnoretin fell well within the predefined limits of acceptability (< or = 15%). After daphnoretin (500 mg/kg) was given orally, the maximum concentration was 0.17 microg/ml at the time of 5 min. The oral bioavailability was about 0.15%.     

Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by 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. Application of liquid chromatography to protein refolding is an exciting step forward for this field. In this work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography (IEC) with a Q Sepharose FF column. Several chromatographic parameters affecting the refolding yield of the denatured/reduced rhG-CSF, such as the urea concentration, pH value, concentration and ratio of reduced/oxidized glutathione in the mobile phase, as well as the flow rate of the mobile phase, were investigated in detail and indicated that the urea concentration and the pH value were of great importance. At the optimal conditions, the renatured and purified rhG-CSF was found to have a specific bioactivity of 3.0 x 10(8) IU/mg, a purity of 96%, and a mass recovery of 49%. Compared with the usual dilution method, the IEC method developed here is more effective for rhG-CSF refolding in terms of specific bioactivity and mass recovery.     

ATP-Responsive Nanoparticles Covered with Biomolecular Machine “Chaperonin GroEL”

Herein, we report an ATP-responsive nanoparticle (^GroELNP) whose surface is fully covered with the biomolecular machine “chaperonin protein GroEL”. ^GroELNP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of ^GroELNP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable ^GroELNP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of ^GroELNP per GroEL was 4.8 and 4.0 times greater than those of precursor ^cysGroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that ^GroELNP could be iteratively extended to double-layered NP.