Periplasmic Inclusion Bodies Research Articles

Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma.

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60% soluble fraction, we obtained a yield of 25mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75% soluble fraction 35mg C7 dAbd per L of cell culture grown in a synthetic media containing 3g/L of vegetable peptone and 1g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.

High-yield expression of human vascular endothelial growth factor VEGF 165 in Escherichia coli and purification for therapeutic applications

Vascular endothelial growth factor (VEGF 165) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9 g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30 ± 6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

In vivo monitoring and alleviation of extracytoplasmic stress to recombinant protein overproduction in the periplasm of Escherichia coli

In Escherichia coli, there are two major pathways, i.e. Cpx and σ E, for dealing with the extracytoplasmic stress in the cell envelope. Due to the unique periplasmic processing steps and the tendency to form periplasmic inclusion bodies, penicillin acylase (PAC) offers a model system for studying the induction of extracytoplasmic stress associated with recombinant proteins overproduction in the periplasm of E. coli. In this study, E. coli strains carrying the lacZ reporter gene fusion with the promoters of three stress-responsive genes, i.e. degP, cpxP, and rpoH, were constructed in the JM109 background for characterization. We demonstrate that pac overexpression induced the extracytoplasmic stress primarily via the Cpx pathway. The upregulated cpxP promoter activity can be a suitable sensor for in vivo monitoring of the extracytoplasmic stress upon pac overexpression. However, such physiological challenge was not observed and all the three promoter activities were reduced when arabinose was used to induce pac overexpression. This result suggests that the physiological impact observed for the IPTG (isopropyl-β- d-thiogalactopyranoside)-induced cultures could be overcome by the use of arabinose for induction. The extracytoplasmic stress response associated with pac overexpression could be significantly alleviated by the exogenous presence of DegP, but only partially alleviated by its mutant derivative of DegP S210A.

Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: A chemical analysis

Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe 3+. Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide.

Preparative expression of secreted proteins in bacteria: status report and future prospects

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.

Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli.

We previously characterized a defective-folding mutant of maltose-binding protein of Escherichia coli, MalE31, which formed periplasmic inclusion bodies. Here, we show that MalE31 aggregation does not affect bacterial growth at 30 degrees C but is lethal at 37 degrees C. Surprisingly, under mild heat shock conditions at 42 degrees C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat shock protease DegP via the CpxA/CpxR two-component signalling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system.

Improving production of penicillin acylase in Escherichia coli via efficient DegP-mediated processing of precursors in periplasm

Penicillin acylase (PAC) from Escherichia coli has a complex enzyme formation mechanism that is unusual for prokaryotic proteins. In this study, PAC is used as a model protein to demonstrate an important concept of developing genetic strategies for improving recombinant protein production in E. coli; namely, the bottleneck gene expression step(s) limiting recombinant protein production must be precisely identified. Using the strong promoter system of trc for regulation of pac gene expression, the overproduction of PAC was often limited by translocation and/or periplasmic processing steps, resulting in intracellular accumulation of various types of PAC precursors. The over-accumulated PAC precursors formed inclusion bodies in the cytoplasm and/or periplasm. The periplasmic protease DegP could efficiently assist periplasmic processing of PAC precursors and, therefore, the amount of periplasmic inclusion bodies was significantly reduced. The PAC inclusion bodies remaining in DegP-coexpressing cells were located in the cytoplasm where the DegP function could not reach. These results indicate that the activity of DegP expression based on pKS12 was high enough for processing the over-accumulated periplasmic PAC precursors in the current expression systems. PAC activity was significantly increased due to DegP-mediated processing in the periplasm. The strategy of DegP coexpression could be further applied to estimate the amounts of various PAC precursors and pac translational efficiency for different pac expression systems could be compared.

DegP-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in Escherichia coli.

We demonstrated the enhancement of recombinant penicillin acylase (PAC) production in Escherichia coli by increasing the intracellular concentration of the periplasmic protease DegP. Using appropriate host/vector systems (e.g., HB101 harboring pTrcKnPAC2902 or MDDeltaP7 harboring pTrcKnPAC2902) in which the expression of the pac gene was regulated by the strong trc promoter, the overproduction of PAC was often limited by periplasmic processing and inclusion bodies composed of protein aggregates of PAC precursors were formed in the periplasm. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The specific PAC activity reached an extremely high level of 674 U/L/OD(600) for MDDeltaP7 harboring pTrcKnPAC2902 and pKS12 under optimum culture conditions. However, such improvement in the production of PAC was not observed for the expression systems (e.g., MDDeltaP7 harboring pCLL2902) in which the periplasmic processing was not the step limiting the production of PAC. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme is shown to be not absolutely required for the formation of active PAC in E. coli. In addition, the steps limiting the production of PAC are identified and the reasons for the formation of PAC inclusion bodies are discussed here.

Degradation versus Aggregation of Misfolded Maltose-binding Protein in the Periplasm of Escherichia coli

The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase.

Various concentrations of isopropyl beta-D-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recombinant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacIq. At low IPTG concentrations (0.025-0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).

Probing the Structural Role of an αβ Loop of Maltose-binding Protein by Mutagenesis: Heat-shock Induction by Loop Variants of the Maltose-binding Protein that Form Periplasmic Inclusion Bodies

The maltose-binding protein (MBP) of Escherichia coliis the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an α/β loop of the structure of MBP that are critical for the in vivofolding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malEwere mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivofolding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon- lacZand htrA- lacZfusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock σ factor, σ 24.

Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli

The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique. The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other beta-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in Escherichia coli using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 degrees C. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.

In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.

Single-chain antibody streptavidin fusions: Tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen

To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabilised the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.

We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5-13% of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

Structure and morphology of protein inclusion bodies in Escherichia coli.

We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence. Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences. Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular. Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain. It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies. Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.