Isolation of inclusion bodies from vegetative Clostridium perfringens: partial purification of a 47 kDa inclusion protein.

A refractile inclusion body produced by vegetative cells of Clostridium perfringens at temperatures above 40 degrees C was isolated and partially characterized. The inclusion was composed of protein and could be solubilized by sodium dodecyl sulphate plus either dithiothreitol or beta-mercaptoethanol. The solubilized inclusion showed no antigenic relationship with Cl. perfringens enterotoxin. One major band with an apparent MW of 47 kDa was demonstrated after polyacrylamide gel electrophoresis of the solubilized inclusion. Both enterotoxin-positive and enterotoxin-negative strains produced the inclusion body. No effect on the morphology of several eucaryotic cell lines was observed when solubilized or intact inclusion was added to the cell cultures.     

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

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

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.     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli.

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers

Overexpression of recombinant N-acetyl-d-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 ± 0.02 U/mg, was similar to that of the native protein, 2.13 ± 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 ± 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 ± 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.     

Effect of inclusion bodies on the buoyant density of recombinant Escherichia coli

Summary During batch culture, buoyant density of recombinant E. coli cells increased linearly as inclusion body per cell increased. This indicated that buoyant density can be used to follow inclusion body formation. This will be helpful to optimize product formation because inclusion bodies are mostly composed of foreign poteins produced by recombinant microorganisms.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

Extraction of rIL-2 inclusion bodies from Escherichia coli using cross-flow filtration

A cross-flow membrane filtration process was developed for the recovery of rIL-2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two-step diafiltration followed by an extraction with 7 M GuHCl and a 40-fold dilution of the solubilized inclusion bodies into 0.01 M Tris-HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL-2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL-2. The membrane process increased the yield of rIL-2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse-phase high-performance liquid chromatography (HPLC). (c) 1994 John Wiley & Sons, Inc.     

Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies

The gene encoding hygromycin B phosphotransferase (hpt) is a widely used selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the non-fusion hpt expression plasmid was constructed and introduced into Escherichia coli to produce enough quantity of the HPT protein. High level expressed HPT was achieved but most of the expressed protein aggregated as inclusion bodies. The inclusion bodies were washed, separated from the cells, and solubilized by 0.3% Sarkosyl. The protein was renatured by dilution and dialysis, and then purified by anion-exchange chromatography. The activity is 8 U/mg protein and the purity is about 95%. Further studies showed that the microbially produced HPT protein had comparable molecular weight, immuno-reactivities, N-terminal amino acid sequences, and biological activities with those of the HPT produced by transgenic rice harboring hpt gene. All these results demonstrated the validity of utilizing the microbially produced HPT to assess the safety of the HPT protein produced in genetically engineered rice.     

Improved solubilization of recombinant human growth hormone inclusion body produced in Escherichia coli

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.     

Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli

The protein composition of inclusion bodies produced in recombinant Escherichia coli overproducing Vitreoscilla hemoglobin (VHb) was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb and pre-beta-lactamase and are separable by differential centrifugation. Results further suggest that the cytoplasmic protein elongation factor Tu is integrated into VHb inclusion bodies. The presence of the outer membrane proteins OmpA and OmpF in inclusion body preparations is attributed to cell envelope contamination rather than specific involvement in inclusion bodies. The specificity of in vivo protein aggregation is discussed.     

表达抗α毒素单链抗体基因工程菌株的构建

将 2种抗A型产气荚膜梭菌α毒素单链抗体 (ScFv)基因ScFv 2E3和ScFv 1A8分别克隆至表达质粒pUC119,pET 2 0b ,pET 2 8a和pHOG2 1中 ,构建了重组质粒 ,分别转化至相应的受体菌JM10 5 ,BL2 1(DE3)和XL1 BLUE中 ,得到表达ScFv的重组菌株。ELISA和SDS PAGE分析检测表明 :经IPTG诱导后所表达的ScFv目的蛋白主要形成了包含体的形式 ,但也有少量ScFv存在于重组菌株的培养上清和胞周质中。经薄层扫描分析 :重组菌株XL1 BLUE (pHOG 2E3)的蛋白表达产物分别占菌体可溶性蛋白的 4% ,重组菌株XL1 BLUE (pHOG 2E3)的蛋白表达产物占菌体总蛋白的相对含量为 2 2 % ,其相对分子量约为 31ku。表达的ScFv蛋白不但具有中和卵磷脂酶的活性 ,而且能够对致死性腹腔攻击α毒素的小鼠产生良好的被动保护作用     

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.     

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.     

A Paracrystalline Inclusion Formed During Sporulation of Enterotoxin-Producing Strains of Clostridium perfringens Type A

A large paracrystalline inclusion is formed by certain strains of Clostridium perfringens type A during spore morphogenesis. In most cell thin sections, the inclusion appeared rod-shaped when sectioned at an angle perpendicular to its longer axis, and circular or oval-shaped when sectioned at an angle parallel to its longer axis. Measurements performed on electron micrographs of inclusions sectioned to reveal the rod shape indicated a fairly consistent thickness (width) of 192 +/- 23 nm. The length of the inclusions varied considerably with a maximum of approximately 2,120 nm being observed. Ultrastructurally, the inclusion was composed of closely packed, periodically spaced, parallel layers. Usually a single inclusion was randomly located in the cytoplasm of the cell. Two inclusions per cell were rarely observed. The inclusion was formed only by ent(+) strains of C. perfringens. Mutants of the ent(+) strain NCTC 8798 that were altered in their sporulating and enterotoxin-producing capacities and revertants of these mutants were tested for inclusion formation. The results indicate that, as with the ent(+) trait, a direct relationship exists between inclusion formation and spore formation. The synthesis of enterotoxin, formation of a morphologically distinct inclusion, and the initial deposition of discontinuous coat fragments around the forespore appear to be events closely related in time during spore morphogenesis.     

Fundamental insights in early-stage inclusion body formation

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.     

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.