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.     

Practical considerations in refolding proteins from inclusion bodies

Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inclusion bodies, (2) change in conformation and flexibility or solubility of proteins during refolding upon reduction of denaturant concentration, and (3) the effect of small molecule additives on refolding and aggregation of the proteins.     

Improved Recovery of a Fusion Protein Containing the Antigenic Domain 1 of the Human Cytomegalovirus Glycoprotein B

Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.     

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.     

The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli

A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, RBBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protein bearing an N-terminal (His)6 purification tag. The expressed recombinant protein, rRBBI-8, is insoluble and accumulates as inclusion bodies. The insoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox conditions. Strategies used to optimize yield and efficiency include selecting the redox system, increasing protein concentration during refolding by adding the denatured protein in a stepwise way, utilizing additives to prevent aggregation, and selecting buffer-exchanging conditions. A Ni-chelate affinity column was then employed to purify the renatured protein. rRBBI-8 shows strong inhibitory activity against trypsin and it can slightly inhibit chymotrypsin. In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system.     

Ultra scale‐down of protein refold screening in microwells: Challenges,solutions and application

Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF‐1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale‐up using hen egg‐white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell‐based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body‐derived protein products. Biotechnol. Bioeng. 2009;103: 329–340. © 2008 Wiley Periodicals, Inc.     

Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies

Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn-HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix-bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength.     

Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.     

包含体蛋白质的复性研究进展

包含体的形成是异源蛋白质在大肠杆菌中高效表达的必然结果,也是目前产生重组蛋白质最有效的方法之一。不可溶、无生物活性的包含体必须经过变性、复性才能获得天然结构,完整特定的生物学功能。聚集是造成重组蛋白质复性产率低下的主要因素,因此理解蛋白质聚集机制,减少和防止聚集的发生是建立高效、高产率复性方法的关键。分子伴侣、低分子量添加物等在复性过程中的应用及新的复性方法的建立都大大提高了重组蛋白质复性产率。     

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.     

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.     

Refolding of protein inclusion bodies directly from E. coli homogenate using expanded bed adsorption chromatography

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.     

Refolding process of cysteine-rich proteins:Chitinase as a model

Background:

Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.

Methods:

Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.

Results:

After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.

Conclusions:

By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.Key Words: Chitinase, Cysteine-rich proteins, Protein refolding, Protein solubilization     

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     

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.     

Analysis of the disulfide bonding pattern between domain sequences of recombinant prochymosin solubilized from inclusion bodies

Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins.Both authors contributed equally to this work     

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

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

A critical assessment of the impact of mixing on dilution refolding

Refolding often presents a bottleneck in the generation of recombinant protein expressed as inclusion bodies. Few studies have looked at the effect of physical factors on the yield from refolding steps. Refold reactors typically operate in fed-batch mode with a slow injection rate. This paper characterizes mixing in a novel reactor, and seeks to relate the conditions of mixing to native lysozyme yields after refolding. A novel twin-impeller system incorporating a mini-paddle impeller located in the vicinity of the injection point was used to increase the local levels of energy dissipation experienced by the injected material, and to improve refolding yields. Mixing only affected yields during and immediately after denatured protein addition. Analysis of lysozyme refolding yield, under a variety of conditions, revealed that dispersive mixing affected the yield. The beneficial effect of the mini-paddle impeller in providing a source of localized energy dissipation was limited to conditions where the bulk impeller intensity was low. The effects appeared to become more significant when injection times were longer, because of increased exposure of the injected material to the energy dissipation of the mini-impeller. The results suggest that for fed-batch protein refolding systems, where mixing has been shown to be a critical factor, the local energy dissipation experienced in the vicinity of the injection point is critical to the refolding yields.