Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.     

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%.     

A comprehensive structure-function analysis shed a new light on molecular mechanism by which a novel smart copolymer, NY-3-1, assists protein refolding

An in-depth understanding of molecular basis by which smart polymers assist protein refolding can lead us to develop a more effective polymer for protein refolding. In this report, to investigate structure-function relationship of pH-sensitive smart polymers, a series of poly(methylacrylic acid (MAc)-acrylic acid (AA))s with different MAc/AA ratios and molecular weights were synthesized and then their abilities in refolding of denatured lysozyme were compared by measuring the lytic activity of the refolded lysozyme. Based on our analysis, there were optimal MAc/AA ratio (44% MAc), M(w) (1700 Da), and copolymer concentration (0.1%, w/v) at which the highest yield of protein refolding was achieved. Fluorescence, circular dichroism, and RP-HPLC analysis reported in this study demonstrated that the presence of P(MAc-AA)s in the refolding buffer significantly improved the refolding yield of denatured lysozyme without affecting the overall structure of the enzyme. Importantly, our bioseparation analysis, together with the analysis of zeta potential and particle size of the copolymer in refolding buffers with different copolymer concentrations, suggested that the polymer provided a negatively charged surface for an electrostatic interaction with the denatured lysozyme molecules and thereby minimized the hydrophobic-prone aggregation of unfolded proteins during the process of refolding.     

Cycloamylose as an efficient artificial chaperone for protein refolding

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase

Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.     

Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: preparation for a functional analysis

Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0M urea in 0.5M increments. Purified RHG1 protein obtained from the 1.5 and 7.0M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0M urea sample, whereas refolding from the 1.5M urea sample yielded 0.2mg/ml protein. The 7.0M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.     

Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.     

Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of some of the denatured proteins. Since identical concentrations of various detergents do not induce similar variations in the protein structures, we arranged to evaluate the artificial chaperoning capabilities of several ionic detergents as a function of charge, structure, and the hydrophobic tail length of the detergent. Our results indicate that carbonic anhydrase can be refolded from its denatured state via artificial chaperone strategy using both anionic and cationic detergents. However, the extent of refolding assistance (kinetic and refolding yield) were different due to protein and detergent net charges, detergent concentrations, and the length of hydrophobic portion of each detergent. These observed differences were attributed to physical properties of CA-detergent complexes and/or to the kinetics of detergent stripping by beta-cyclodextrin from the protein-detergent complexes which is apparently dependent on the detergent-beta-CD association constants and the nature of the partially stripped complexes.     

Oxidative refolding chromatography: folding of the scorpion toxin Cn5

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.     

Refolding of protein inclusion bodies directly from E. coli homogenate using expanded bed adsorption chromatography

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Refolding of the full-length non-structural protein 3 of hepatitis C virus

An easy and reproducible procedure for purification and refolding of the full-length non-structural protein 3 (NS3) from hepatitis C virus has been developed. Refolding was achieved by simply diluting the protein into a suitable buffer. Low protein concentration, high pH, highly reducing conditions, the presence of detergent, and low viscosity were important parameters for high refolding efficiency. Refolding was insignificantly affected by the presence of Zn(2+) in the refolding buffer, while the addition of NS4A cofactor inhibited refolding. A comparison of the kinetic parameters showed that the refolded enzyme is not as catalytically competent as the native enzyme. Nevertheless, the activity of the refolded NS3 protease was dependent on the specific NS4A-peptide cofactor and was inhibited by the specific substrate-based NS3 protease inhibitor, which indicates that the refolded NS3 can be appropriate for inhibitor screening. The yield of pure protein from the insoluble fraction of cell lysate was 6 mg/L of bacterial culture, which is 18 times higher than obtained from the soluble fraction. Improvement of the refolding conditions has resulted in a 50-fold higher activity of the protease as compared to refolding in buffer with neutral pH and no additives.     

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

Production of correctly folded and biologically active proteins in Escherichia coli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS–PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from 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.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

Evaluation of Chaperone-like Activity of Alginate: Microcapsule and Water-soluble Forms

To evaluate the chaperone-like activity of alginate stabilization and refolding of alkaline phosphatase (ALP) was investigated in the presence of alginate through two different approaches, the soluble form and microcapsule assisted methods. It was found that in the presence of microcapsules, ALP can be stabilized to a higher degree compared with the water-soluble form, whereas the denatured ALP is refolded with a higher yield through latter method. Lower refolding yields of alginate beads compared with its soluble form may be the result of lower refolding rate of ALP upon elution of the bound enzyme by dispersing the precipitate in NaCl which left the unfolded protein in an unsuitable environment, providing enough time for protein aggregation and leading finally to lower recovered activity compared with application of soluble form of alginate. In addition in the case of alginate capsules, the choice of suitable divalent ion is essential for stability and assistance in refolding.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.