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.     

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.     

Affinity purification of recombinant interferon-alpha on a mimetic ligand adsorbent

A method for improved refolding and purification of recombinant human interferon-alpha (rh-IFN-alpha) from inclusion bodies is described. The optimal conditions of refolding were obtained by the addition of 0.5 M l-arginine to the refolding buffer. The rh-IFN-alpha was purified to near homogeneity utilizing a single-step chromatography on a mimetic dye-ligand matrix. Improved refolding, coupled to a single-column affinity purification strategy, resulted in a 10-fold increase in the yield of rh-IFN-alpha. This single-step purification protocol yielded approximately 50 mg of purified rh-IFN-alpha from 1 liter of shake flask culture. The rh-IFN-alpha prepared by this protocol was found to be essentially monomeric based on HPLC gel filtration and nonreducing SDS-PAGE. It had a specific activity of approximately 2.8 x 10(8) IU/mg, measured as inhibition of cytopathic effect of encephalomyocarditis virus on A549 human lung carcinoma cells.     

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.     

In vitro activation, purification, and characterization of Escherichia coli expressed aryl-alcohol oxidase, a unique H2O2-producing enzyme

Aryl-alcohol oxidase (AAO), a flavoenzyme with unique spectral and catalytic properties that provides H2O2 for fungal degradation of lignin, has been successfully activated in vitro after Escherichia coli expression. The recombinant AAO (AAO*) protein was recovered from inclusion bodies of E. coli W3110 transformed with pFLAG1 containing the aao cDNA from Pleurotus eryngii. Optimization of in vitro refolding yielded 75% active enzyme after incubation of AAO* protein (10 microg/ml) for 80 h (at 16 degrees C and pH 9) in the presence of glycerol (35%), urea (0.6 M), glutathione (GSSG/GSH molar ratio of 2), and FAD (0.08 mM). For large-scale production, the refolding volume was 15-fold reduced and over 45 mg of pure active AAO* was obtained per liter of E. coli culture after a single anion-exchange chromatographic step. Correct FAD binding and enzyme conformation were verified by UV-visible spectroscopy and circular dichroism. Although the three enzymes oxidized the same aromatic and aliphatic polyunsaturated primary alcohols, some differences in physicochemical properties, including lower pH and thermal stability, were observed when the activated enzyme was compared with fungal AAO from P. eryngii (wild enzyme) and Emericella nidulans (recombinant enzyme), which are probably related to the absence of glycosylation in the E. coli expressed AAO.     

Overproduction and characterization of xylanase B from Aspergillus niger

The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.     

Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase

The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.     

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens

The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.     

Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma

The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth. In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.     

On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment

The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.     

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.     

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%.     

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

One-step Purification and Immobilization in Cellulose of the GroEL Apical Domain Fused to a Carbohydrate-binding Module and its use in Protein Refolding

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD_Cex, of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.     

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies

The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.     

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.