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%.     

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.     

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.     

重组羧肽酶原B的复性方法研究

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni2 亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni2 亲合层析>透析复性。     

Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.

Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.     

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25 column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding. Revisions requested 31 October 2005; Revisions received 20 December 2005     

Preparative protein refolding

The rapid provision of purified native protein underpins both structural biology and the development of new biopharmaceuticals. The dominance of Escherichia coli as a cellular biofactory depends on technology for solubilizing and refolding proteins that are expressed as insoluble inclusion bodies. Such technology must be scale invariant, easily automated, generic for a broad range of similar proteins and economical. Refolding methods relying on denaturant dilution and column-based approaches meet these criteria. Recent developments, particularly in column-based methods, promise to extend the range of proteins that can be refolded successfully. Developments in preparing denatured purified protein and in the analysis of protein refolding products promise to remove bottlenecks in the overall process. Combined, these developments promise to facilitate the rapid and automated determination of appropriate refolding conditions and to simplify scale-up.     

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.     

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.     

重组抗血红素ScFv包涵体蛋白的复性及纯化研究

抗血红素多二硫键ScFv在大肠杆菌中绝大多数表达产物为包涵体,为了获得可溶性的具有生物活性的ScFv,摸索了不同的复性条件,包括透析法、稀释和层析相结合的方法。研究发现,先对溶解的变性ScFv溶液稀释,进行初步的蛋白质复性,再利用Sephadex G-25凝胶层析进一步复性、降低变性剂浓度和纯化,至少可以得到95％纯度,产率为150mg／L的目标蛋白,通过一次凝胶过滤层析,达到了去除变性剂、复性及纯化ScFv蛋白三种目的,为多二硫键ScFv在大肠杆菌中的表达和纯化提供了一种经济可行的方法。     

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.     

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.     

A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient

Summary A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 10⁵ IU mg⁻¹and active protein recovery was up to 54%.     

Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3

Recombinant Arabidopsis thaliana (At) RGL-3, using two vectors pMAL-c2 and pET 21, was expressed as inclusion bodies in Escherichia coli under a range of temperature conditions. Only low levels (8-12% of total protein) of soluble protein were produced. The "soluble" fraction was shown by native PAGE to exist as soluble aggregates of RGL-3. A method was developed, consisting of induction of expression at various temperatures that yielded high levels of refoldable inclusion bodies using the pET vector. (At) RGL-3, as inclusion bodies, was solubilized in 8M urea and refolding was initiated by 20-fold direct dilution of denaturant. Under optimal conditions, 87% of the denatured protein of inclusion bodies was successfully re-natured. Refolding was monitored by "native" PAGE. Refolded RGL-3 was shown to be present as monomers and dimers. Attempts to further purify His-tagged RGL-3 using Ni/NTA chromatography resulted in the formation of higher polymers.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Isolation of human interferon β1b (Ser17) expressed in <Emphasis Type="Italic">E. coli</Emphasis> with the use of ion-exchange chromatography

A method for isolation of interferon β1b (Ser17) from the inclusion bodies is presented. This method consists of the following stages: solution and reduction of the protein from the inclusion bodies, refolding, chromatography on DEAE-sepharose, chromatography on SP-sepharose, concentration of the protein solution, demineralization, and addition of stabilizers. The refolding was performed by dilution of the solution of the reduced protein with a buffer (pH 8.0) containing 50 mM Tris-HCl, 25 μM CuCl₂, and 0.5% Tween-20. Interferon β1b was eluted from a cation-exchange column with a pH gradient (9.3–11.3) of sodium phosphate buffer. The eluate was concentrated and desalted on a column with sephadex G-50 in 1 mM NaOH, mannitol, and sodium phosphate were added for neutralization of the protein solution. The product was homogenous according to gel electrophoresis and HPLC, and exhibited the practically same antiproliferative activity as that of Betaferon taken as a standard. Thus, a possibility of preparation of the purified and active interferon β by ion-exchange chromatography in the presence of the Tween-20 nonionic detergent was demonstrated. The proposed procedure is promising for application to large-scale production and industry.     

Isolation of expressed in E. coli human interferon beta1b (Ser17) by ion-exchange chromatography

A method for isolation of interferon beta1b (Serl7) from inclusion bodies, comprising the steps of solution and reduction of protein from the inclusion bodies, refolding, chromatography on DEAE-Sepharose, chromatography on SP-Sepharose, concentrating, desalting and addition of stabilizers. The solution of reduced protein was diluted with pH 8.0 buffer of 50 mM Tris-HCl, 25 microM CuCl2 and 0.5% Twin 20 for refolding. We used gradient of pH (from 9.3 upto 11.3) for elution of interferon-beta from cation-exchange column. We concentrated of eluate and then desalted on the Sephadex G-50 column with 1 mM NaOH. Then the protein solution was neutralized with mannitol and Na-phosphate. Obtained preparation of interferon-beta was pure by gel-electrophoresis and by HPLC analysis, and had practically indentical level of antiproliferative activity with well-known preparation of Betaferone. Thus we show the possibility of isolation and obtaining of pure and active interferone-beta by ion-exchange chromatography in the presence of non-ion detergent Twin 20. We believe this method for interferon betalb preparation is perspective for scaling and using in the develop of industrial technology for production of this preparation.     

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.     

Recombinant Human Ribosomal Protein S16: Expression,Purification, Refolding,and Structural Stability

The cDNA of human ribosomal protein S16 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. It was found that 21 +/- 4% of the amino acid sequence of the protein forms alpha-helices and 24 +/- 3% is in beta-strands. The protein structure stability was studied at various pH values and urea concentrations. The protein is quickly denatured at pH above 8.0, whereas increasing of urea concentration causes slow unfolding of the protein.     

Renaturation of human proinsulin--a study on refolding and conversion to insulin

The production of human proinsulin in Escherichia coli usually leads to the formation of inclusion bodies. As a consequence, the recombinant protein must be isolated, refolded under suitable redox conditions, and enzymatically converted to the biologically active insulin. In this study we describe a detailed in vitro renaturation protocol for human proinsulin that includes native structure formation and the enzymatic conversion to mature insulin. We used a His(8)-Arg-proinsulin that was renatured from the completely reduced and denatured state in the presence of a cysteine/cystine redox couple. The refolding process was completed after 10-30 min and was shown to be strongly dependent on the redox potential and the pH value, but not on the temperature. Refolding yields of 60-70% could be obtained even at high concentrations of denaturant (3M guanidinium-HCl or 4M urea) and protein concentrations of 0.5mg/ml. By stepwise renaturation a concentration of about 6 mg/ml of native proinsulin was achieved. The refolded proinsulin was correctly disulfide-bonded and native and monomeric as shown by RP-HPLC, ELISA, circular dichroism, and analytical gel filtration. Treatment of the renatured proinsulin with trypsin and carboxypeptidase B yielded mature insulin.