Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent

A method for the production and purification of biologically active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), expressed in Escherichia coli, has been developed. Washing of an inclusion body fraction which allows the removal of numerous ballast proteins and on-column refolding of rhGM-CSF are specific characteristics of the method. The refolding efficiency reached 70%; the purity of the preparation constituted 95%. The biological activity was similar to those of other recombinant hGM-CSF analogs.     

大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）高效表达克隆ｐＺＷ．ＧＭ的表达产物进行了纯化,并对纯化的ＧＭ－ＣＳＦ进行了Ｎ端氨基酸序列分析。人ＧＭ－ＣＳＦ基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过超声破菌、包涵体抽提、凝胶过滤层析、复性、离子交换一系列化步骤,终产物纯度达９９％,按蛋白总量计算回收率达１０％,比活性达１×１０＾７ｕ／ｍｇ蛋白质。通过测定纯化人ＧＭ－ＣＳＦ的Ｎ端１     

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.     

Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli

To renature the inactive rhBMP-2 which overexpressed in Escherichia coli, post-expression treatments including inclusion bodies solubilization and in vitro refolding were systematically investigated. An optimized refolding process was established from screening and successfully scaled up with yield greater than 70%. Then, hydrophobic interaction chromatography (HIC) was adopted as two consecutive stages to separate the active rhBMP-2 homodimer from refolding mixture. Aiding additive N,N-dimethylformamide (DMF) was found to enhance the resolution of rhBMP-2 homodimer most effectively. The rhBMP-2 homodimer was purified to homogeneity through two HIC separations at different salt contents, the purified rhBMP-2 homodimer was fully bioactive and had equivalent biological activity to rhBMP-2 produced from Chinese hamster ovary cell (CHO). Under the optimal refolding and purification conditions, 80 mg rhBMP-2 homodimer with high purity could be obtained from 1 g wet weight of inclusion bodies. Finally, this efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant.     

An Automatic Refolding Apparatus for Preparative-Scale Protein Production

Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.     

Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1

G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the β1 and β2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli.     

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6–8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.     

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。     

Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.     

Co-Solvents as Stabilizing Agents during Heterologous Overexpression in Escherichia coli – Application to Chlamydial Penicillin-Binding Protein 6

Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the “chlamydial anomaly.”     

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.     

A screening system for the identification of refolding conditions for a model protein kinase, p38alpha

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38α, and applied this system to denatured p38α derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38α can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38α protein kinase.     

Effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor (Cys17Ser) in aqueous two-phase systems containing chelated metal ions

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.     

Improved Production of the Human Granulocyte-Macrophage Colony Stimulating Factor in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds Using a Dual Sorting Signal Peptide

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) containing either an endoplasmic reticulum (ER) retention signal or a phaseolin vacuolar sorting signal peptide was expressed in Arabidopsis thaliana under the control of a tissue-specific promoter, derived from the soybean α′ subunit of β-conglycinin. No significant differences in recombinant hGM-CSF (rhGM-CSF) accumulation were detected between transgenic plants carrying either one of the two signal peptides. Hybrid seed from crosses between single-copy transformants tailed with the ER retention signal tetrapeptide and single-copy transformed plants tagged with a phaseolin four carboxy-terminal residues showed gene additive effects. The highest expression level of rhGM-CSF was 0.05% of total soluble protein of immature siliques, indicating that the two signal peptides functioned independently in the protein-sorting pathway. Additionally, TF-1 cell proliferation data demonstrated that rhGM-CSF was biologically active.     

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.     

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%.     

Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high‐efficiency protein refolding. We have herein found that electrostatic repulsion between like‐charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion‐exchange resins when the protein and ion‐exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like‐charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like‐charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.     

A suitable and effective stepwise oxidative refolding procedure for highly-cationic tetrameric avidin in nucleic acid free conditions

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.