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.     

A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies.

Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL. This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL). This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8616-8620). Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E. coli in inclusion bodies and must be denatured and refolded to become active. This requires correct folding, formation of native disulfide bonds, and the association of different domains. All these steps are strongly dependent on the renaturation conditions used. Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as L-arginine. Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution. This approach should be useful for the production of active forms of other recombinant proteins.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.     

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.     

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.     

Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli

This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni(2+)) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.     

Oxidative renaturation of lysozyme at high concentrations

Newly synthesized cloned gene proteins expressed in bacteria frequently accumulate in insoluble aggregates or inclusion bodies. Active protein can be recovered by solubilization of inclusion bodies followed by renaturation of the solubilized (unfolded) protein. The recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process is generally conducted at low protein concentrations (0.01-0.2 mg/mL) to avoid aggregation. We have investigated the potential of successfully refolding reduced and denatured hen egg white lysozyme at high concentrations (1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically favor proper folding over inactivation were found. Solubilizing agents such as guanidinium chloride (GdmCl) and folding aids such as L-arginine present in low concentrations during refolding effectively enhanced renaturation yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic competition between lysozyme folding and aggregation can be described using first-order kinetics for the renaturation reaction and third-order kinetics for the overall aggregation pathway. The rate constants for both reactions have been found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necessity to reactivate proteins at low concentrations using large renaturation volumes. The marked increase in volumetric productivity makes this a viable option for recovering biologically active protein efficiently and in high yield in vitro from proteins produced as inclusion bodies within microbial cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 221-230, 1997.     

Purification and characterization of recombinant extracellular domain of human HER2 from Escherichia coli

The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family, and it plays an important role in the development of many human adenocarcinomas. The extracellular domain (ECD) of HER2 is an ideal target for therapeutic approaches. In order to obtain large quantities of active HER2 ECD protein for biochemical and structural analysis and for detecting anti-HER2 ECD antibodies in serum, a systematic assessment of optimal parameters for the refolding of the glutathione S-transferase (GST) fusion protein was carried out. After the GST-HER2 ECD inclusion bodies were solubilized with denaturation buffer containing 8M urea, an approach was then used to optimize refolding parameters. This approach utilized dilution of denatured and reduced GST-HER2 ECD into different refolding buffers using orthogonal design method. Optimal refolding was obtained in an alkaline buffer containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for 24h. After purification with glutathione Sepharose 4B and PreScission protease cleavage of the fusion protein, 8.9mg of recombinant HER2 ECD was obtained from 1L of Escherichia coli. Rabbit polyclonal antibodies against HER2 ECD were obtained. The purified protein was found to be immunogenic and useful for immunodiagnostic studies of serum HER2 ECD and its antibodies by using enzyme-linked immunosorbent assay (ELISA).     

Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2

The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2.     

重组人白细胞介素4的纯化及N端氨基酸序列分析

本文对重组人白细胞介素4高效表达克隆pBV220/hIL-4a的表达产物进行了纯化,升对纯化的人IL-4进行了N端氨基酸序列分析。人IL-4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、复性浓缩、离子交換和凝胶过滤层析一系列纯化步骤,终产物纯度达98％以上,按蛋白总量计算回收率为14％,比活性达2×10~6单位/mg蛋白。通过测定纯化人IL-4的N端16个氮基酸序列,与由其DNA序列推导的氨基酸序列完全一致。本文为重组人IL-4的批量生产奠定了基础。     

The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.     

Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

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.     

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。     

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.     

大肠杆菌异源重组蛋白质的变性与复性

描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。     

Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli

We have cloned, overexpressed, and purified the catalytic domain (residues Gly106 to Asn268) of human macrophage metalloelastase (MMP-12) in Escherichia coli. This construct represents a truncated form of the enzyme, lacking the N-terminal propeptide domain and the C-terminal hemopexin-like domain. The overexpressed protein was localized exclusively to insoluble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclusion bodies were solubilized in an 8 M guanidine-HCl buffer and purified by gel filtration chromatography under denaturing conditions. Partial refolding of the protein by dialysis into a 3 M urea buffer caused selective degradation of the truncated form of the protein, while the intact catalytic domain was unaffected by proteolysis. An SP-Sepharose chromatography step purified the protein to homogeneity and served also to complete the refolding. The purified protein was homogeneous by mass spectrometry and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a concentration of 9 mg/ml. This purification procedure enables the production of 23 mg of protein per liter of E. coli culture and is amenable to large-scale protein production for structural studies.     

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。