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%.     

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.     

Characterisation of the dominant oxidative folding intermediate of hen lysozyme.

Reduced denatured lysozyme has been oxidised and refolded at pH values close to neutral in an efficient way by dilution from buffers containing 8.0 M urea, and refolding intermediates were separated by reverse-phase HPLC at pH 2. By using peptic digestion in combination with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem MS/MS the dominant intermediate was identified to be des-[76-94]. This species has three of the four native disulphide bonds, but lacks the Cys76-Cys94 disulphide bond which connects the two folding domains in the native protein. Characterisation of des-[76-94] by 2D1H NMR shows that it has a highly native-like structure. This provides an explanation for the accumulation of this species during refolding as direct oxidation to the fully native protein will be restricted by the burial of Cys94 in the protein interior.     

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.     

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.     

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     

层析辅助体外复性含有多对二硫键的融合蛋白质

为建立一种基于阴离子交换介质辅助的含多对二硫键的抗凝溶栓双功能水蛭素12肽-瑞替普酶融合蛋白质 (HV12p-rPA) 的复性方法,采用Q Sepharose XL作为层析复性介质,通过正交实验考察蛋白质上样量、流速、脲梯度、洗脱液中精氨酸浓度、脲浓度、pH、还原型及氧化型谷胱甘肽等因素对复性过程的影响,探索最佳层析复性条件。结果表明：脲梯度、上样量及精氨酸浓度是影响复性的3个主要因素。脲梯度是复性成功的关键,上样量增大时复性蛋白质比活降低,精氨酸辅助HV12p-rPA复性的最佳浓度为1 mol/L。创建了脲、pH双梯度下的阴离子交换层析辅助HV12p-rPA的复性方法,复性后蛋白质的溶栓比活达到46 520 IU/mg,抗凝比活达到9 980 ATU/mg,与稀释复性方法相比,该方法能使复性蛋白质的溶栓比活提高14~15倍,抗凝比活提高7~8倍。     

High‐throughput protein refolding screening method using zeolite

We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Renaturation and activation of calf prochymosin produced in an insoluble form in Escherichia coli

Calf prochymosin produced in Escherichia coli cells harboring the expression plasmids was insoluble and formed large inclusion bodies, which were solubilized by 8 M urea. The conditions allowing correct refolding of denatured prochymosin were investigated. Dialysis at pH 10 in the presence of 500 mM NaCl was found to give the maximum renaturation, and subsequent acidic treatment for autocatalytic processing of refolded prochymosin allowed almost 100% recovery of chymosin.     

Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.     

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.     

pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.     

Single pH buffer refolding screen for protein from inclusion bodies

We previously reported the set up of an automated test for screening the refolding of recombinant proteins expressed as inclusion bodies in Escherichia coli[1]. The screen used 96 refolding buffers and was validated with 24 proteins, 70% of which remained soluble in at least one buffer. In the present paper, we have analyzed in more detail these experimental data to see if the refolding process can be driven by general rules. Notably, we found that proteins with an acidic isoelectric point (pI) refolded in buffers the average pH of which was alkaline and conversely. In addition, the number of refolding buffers wherein a protein remained soluble increased with the difference between its pI and the average pH of the buffers in which it refolded. A trend analysis of the other variables (ionic strength, detergents, etc.) was also performed. On the basis of this analysis, we devised and validated a new refolding screen made of a single buffer for acidic proteins and a single buffer for alkaline proteins.     

尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ , 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化, 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论     

Alginate-chaperoned facile refolding of Chromobacterium viscosum lipase

Urea denatured lipase from Chromobacterium viscosum lipase could be refolded by addition of alginate with high guluronic acid content. The refolded molecule could be recovered by affinity precipitation. This approach resulted in recovery of 80% (of original activity) as compared to classical dilution method which gave only 21% activity recovery. Dynamic light scattering showed that binding required about 45 min and activity data obtained from affinity precipitation experiments indicated that refolding was almost instantaneous after binding. Circular dichroism (CD) and fluorescence data showed that refolded molecule was identical to the native molecule. It also showed that refolding takes place at the binding stage and not at the precipitation stage. Preliminary studies showed that the refolding strategy worked equally well with lipases from wheat germ and porcine pancreas.     

Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A2 (A .aBPLA2 )基因克隆至温敏表达载体 pBLMVL2 ,在大肠杆菌RR1中成功诱导表达 .表达产物A .aBPLA2 约占细菌蛋白质总量的 2 0 % ,并以包涵体的形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用FPLCSuperoseTM12纯化 ,产物经过SDS 聚丙烯酰胺凝胶电泳检测只有单一条带 .对纯化后的表达A .aBPLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,表达A .aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2 酶活性相近 ,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2 的溶血活性 ,没有抑制血小板聚集活性 .最后对磷脂酶A2 的结构与这些活性的关系进行了讨论     

Refolding of superoxide dismutase by ion-exchange chromatography

A new ion-exchange chromatography process was developed for refolding of iron superoxide dismutase (Fe-SOD) produced in Escherichia coli as an inclusion body. After adsorption on an ion-exchange matrix, the denatured protein was eluted by gradient decrease of urea concentration and pH of the elution buffer. The dual gradient allowed the denatured protein to refold to its correct native conformation with return of biological activity. Compared with the traditional dilution, refolding process, the new process increased the refolding yield five-fold. The process could also be carried out at high protein concentration to decrease the solution volume after refolding.     

Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida

Pasteurella multocida toxin is a potent mitogen that is believed to act intracellularly. On transverse urea gradient gels at pH 8.0 the toxin displayed one major unfolding transition at 4 M urea. However, at pH 6.1 the unfolding transition took place at 3.5 M urea. Circular dichroism spectra also indicated that a structural change took place at acidic pH. In addition it was found that the toxin that had been denatured in 8 M urea refolded in solution with a high recovery of biological activity. These findings are discussed in terms of the likely domain structure of the P. multocida toxin.     

On-column refolding of recombinant human interferon-gamma inclusion bodies by expanded bed adsorption chromatography

A refolding strategy was described for on-column refolding of recombinant human interferon-gamma (rhIFN-gamma) inclusion bodies by expanded bed adsorption (EBA) chromatography. After the denatured rhIFN-gamma protein bound onto the cation exchanger of STREAMLINE SP, the refolding process was performed in expanded bed by gradually decreasing the concentration of urea in the buffer and the refolded rhIFN-gamma protein was recovered by the elution in packed bed mode. It was demonstrated that the denatured rhIFN-gamma protein could be efficiently refolded by this method with high yield. Under appropriate experimental conditions, the protein yield and specific activity of rhIFN-gamma was up to 52.7% and 8.18 x 10(6) IU/mg, respectively.