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.     

Preparative method for obtaining recombinant human interferon α2b from inclusion bodies of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A simple, easily reproducible, and scalable method for obtaining recombinant human interferon α2b from Escherichia coli inclusion bodies has been elaborated. It involves the following steps: preparation of producer cell biomass, isolation and washing of inclusion bodies, their dissolution with protein refolding, SP Sepharose chromatography, and DEAE Sepharose chromatography. According to the results of gel electrophoresis and reversed-phase HPLC, the purity of the protein obtained exceeds 95%.     

Chromatographic methods for the isolation of,and refolding of proteins from,Escherichia coli inclusion bodies

New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies.     

High throughput purification of recombinant human growth hormone using radial flow chromatography

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 μm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

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.     

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.     

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.     

Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

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     

Refolding of fusion ferritin by gel filtration chromatography (GFC)

Fusion ferritin (heavy chain ferritin, F_H+light chain ferritin, F_L), an iron-binding protein, was primarily purified from recombinantEscherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, andl-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclussion bodies (Ibs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.     

Purification, Refolding of Hybrid hIFNγ-Kringle 5 Expressed in Escherichia coli

The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon gamma and over-produced in the form of inactive inclusion bodies in E. coli. The amount of fusion protein was about 40% of total protein produced. The fusion protein contained in the inclusion bodies was solubilized in 8 m urea and purified by anion-exchange chromatography. We employed the orthogonal experimental design L₁₆(4⁵) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor l-arginine, NaCl, pH, and glycine on the refolding procedure. Our results suggest that the presence of appropriate l-arginine, NaCl, and denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein with biological activity. The refolded fusion protein, γIFN/pk5, has in vitro anti-endothelial cell proliferation activity. Received: 24 July 2000 / Accepted: 21 September 2000     

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。     

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.     

Production,IMAC purification,and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide

A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified. The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG. Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins.     

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.     

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.