Modeling of protein refolding from inclusion bodies

Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies （IBs）, which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble pro- teins following dilution refoiding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical proper- ties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high effi- cacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight （R2= 0.98, P = 0.000, F-test）. Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refold- ing developed here could be used to predict the refold- ing yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refoiding from IBs according to their intrinsic properties.     

Recovery of functionally‐active protein from inclusion bodies using a thermal‐cycling method

Heterologous overexpression of genes in Escherichia coli has made it possible to obtain high titers of recombinant proteins. However, this can result in the formation of aggregated protein particles known as ‘inclusion bodies’. Protein sequestered as inclusion body is inactive and needs to be converted back to its functional form by refolding using appropriate techniques. In the current study inclusion bodies of the enzyme aminoglycoside nucleotidyl transferase (or ANT(2″)‐Ia) were first solubilized in urea and subsequently subjected to thermal cycling under controlled conditions as part of the refolding strategy. Thermal cycling led to disaggregation of the individual protein chains and simultaneously refolding the released protein molecules to their native state. The optimum condition was identified as 10–80°C thermal cycling at 3°C s^?1 for 2 h. Enzyme activity measurements showed that thermal cycling under optimized conditions resulted in 257% activity recovery when compared with nonrefolded protein. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:133–139, 2017     

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.     

Refolding of porcine growth hormone from inclusion bodies of Escherichia coli

Escherichia. coli cells expressing porcine growth hormone were grown in a batch fermentation process. The expression level was estimated to be nearly 40% of the total cellular protein after 2–3 h of induction with 1?mM isopropyl β-d-thiogalactoside. Porcine growth hormone expressed as inclusion bodies was solubilized in 8 M urea. Refolding conditions following a dilution protocol in the presence of β-mercaptoethanol or using a glutathione pair were tested. Reverse phase-HPLC was applied to distinguish oxidized, misfolded and reduced forms of the hormone. A ratio of reduced to oxidized glutathione equal to 2/1 was chosen to avoid the formation of misfolded forms at high protein concentration.     

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.     

Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.     

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 strategies from inclusion bodies in a structural genomics project

The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.     

A simplified method for the efficient purification and refolding of recombinant human TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to the TNF cytokine superfamily that specifically induces apoptosis in a broad spectrum of human cancer cell lines but not in most healthy cells. The antitumor potential of recombinant human TRAIL (rhTRAIL) has attracted great attention among biologists and oncologists. However, attempts to express rhTRAIL in Escherichia coli often results in limited yield of bioactive protein due to the formation of inclusion bodies (IBs), which are dense insoluble particulate protein aggregates inside cells. We describe herein a highly simplified method to produce pure bioactive rhTRAIL using E. coli. The method is straightforward and requires only basic laboratory equipment, with highly efficient purification and high yield of renaturation, and may also be applied to produce other proteins that form IBs in E. coli.     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

Solubilization and regeneration of Vitreoscilla hemoglobin isolated from protein inclusion bodies

Vitreoscilla hemoglobin (VHb), a homodimeric protein containing two heme groups in its native state, was used as a model to investigate inclusion body approtein solubilization, prosthetic group incorporation, and reactivation. High-level expression in recombinant Escherichia coli results in accumulation of a substantial portion of heme-free VHb in inclusion bodies. VHb can be solubilized from these inclusion bodies by relatively low concentrations of urea with the dissolution midpoint at approximately 3.2M urea. Dissolution in the presence of stoichiometric heme shifts the dissolution midpoint to approximately 4.5M urea without influencing the dissolution properties of contaminant proteins, suggesting the effect is specific for VHb. Denaturation of apoVHb and holoVHb obtained from purified native VHb has midpoints of 2.9M and 5.1M urea, respectively. VHb solubilized from inclusion bodies with urea at concentrations from 0 to 3.5M urea can be regenerated by heme addition without dilution of urea to yield active holoVHb. The fraction of solubilized VHb reconstituted upon heme addition is maximum at around 30% when solubilization and reconstitution is conducted in less than 1M urea. At these low urea concentrations, approximately 5% of inclusion body VHb is solubilized. These results show the utility of prosthetic group addition to reconstitute holoVHb in the presence of urea. Also, these findings suggest that some inclusion body protein has partially folded conformation and that a fractional dissolution and refolding process may be advantageous.     

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     

重组蛋白复性技术研究进展

本对近年来重组蛋白复性技术的研究进行了评述。比较分析了液相和固相复性的各种方法,提出了复性优化的方案,介绍了在化学复性基础上发展物理复性如高压复性法的新思路。     

Aggresomes, inclusion bodies and protein aggregation

Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.     

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

Expression and purification of aggregation‐prone and disulfide‐containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high‐value target proteins. Here, we present a novel gene‐fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag‐Acidic‐Target Tag) results in near‐complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one‐step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.     

Coupling of chemical extraction and expanded-bed adsorption for simplified inclusion-body processing: optimization using surface plasmon resonance

Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated. The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein. Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation. However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated. The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR). The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction. The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA. The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery. The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%). This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies.     

High-pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: refolding optimization, and feasibility assessment

High-pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1-3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large-scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29-42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high-pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high-pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale-up for this molecule.     

Recombinant murine growth hormone from E. coli inclusion bodies: Expression,high‐pressure solubilization and refolding,and characterization of activity and structure

We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.