Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.

Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)(260)GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the P(BAD) promoter. The expression system based on the P(BAD) promoter was found to be superior to the T7-based system.     

Effect of tumor necrosis factor on acetyl-coenzyme A carboxylase gene expression and preadipocyte differentiation

Tumor necrosis factor (TNF) is secreted by macrophages in response to various stimuli and blocks lipid accumulation during the conversion of preadipocytes to adipocytes in culture. In the present report, we investigate the effect of recombinant TNF on the expression of acetyl-coenzyme-A (CoA) carboxylase, the rate-limiting enzyme for long-chain fatty acid biosynthesis. We used a preadipocyte cell line, 30A-5, derived from 10T1/2 mouse fibroblasts after treatment with 5-azacytidine. Treatment of the preadipocyte cell line with dexamethasone and insulin triggers the conversion of these cells to mature adipocytes as evidenced by the accumulation of lipid. The mRNA and enzyme levels of acetyl-CoA carboxylase as well as the enzyme activity increase markedly during the conversion process. TNF prevents the conversion of preadipocytes to adipocytes with a concomitant inhibition in the accumulation of acetyl-CoA carboxylase mRNA and decrease in enzyme activity. This observed reduction in acetyl-CoA carboxylase mRNA levels is reversible upon removal of TNF. Acetyl-CoA carboxylase mRNA levels and enzyme activity also decrease when fully differentiated adipocytes are exposed to TNF but to a much lesser extent. These results suggest that TNF affects de novo lipid synthesis in part by altering the mRNA levels of acetyl-CoA carboxylase.     

Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins

High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.     

Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts

Human endothelial cells and dermal fibroblasts both expressed a membrane-associated interleukin 1 (IL-1) activity when stimulated with either recombinant tumor necrosis factor (TNF) or recombinant lymphotoxin but stimulated endothelial cells expressed significantly more membrane IL-1 per cell than did fibroblasts. Lipopolysaccharide induced membrane IL-1 activity on endothelial cells but not fibroblasts. Interferon-gamma treatment of endothelial cells and fibroblasts had no direct effect on membrane IL-1 expression and little effect when used as a pretreatment for TNF or lipopolysaccharide stimulation. Endothelial cell membrane IL-1 activity was induced within 24 hr of culture with TNF or lipopolysaccharide, and increased up to 72 hr of incubation. Antibodies raised against human monocyte-derived IL-1 species neutralized the membrane IL-1 activity of TNF-stimulated endothelial cells. Both absorption studies and neutralization with specific sera indicated that endothelial cell membrane IL-1 is structurally related to IL-1 alpha. Endothelial cells expressed both IL-1 beta mRNA in response to TNF, lymphotoxin, and recombinant IL-1 species, as detected by Northern blot analysis. These studies demonstrate that endothelial cells can be activated to express a cell-surface IL-1 activity which is structurally, as well as functionally, related to the secreted form of IL-1.     

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.     

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.     

The use of nonlinear dielectric spectroscopy to monitor the bioelectromagnetic effects of a weak pulsed magnetic field in real time

Nonlinear dielectric spectroscopy (NLDS) was used to detect interaction of a pulsed magnetic field (PMF) with membrane protein dynamics in aggregating Dictyostelium discoideum amoebae. In the experiments reported here, a strong nonlinear dielectric response of Dictyostelium discoideum cells is shown, and a distinctive nonlinear dielectric response of cells previously exposed to PMF is shown. The method of NLDS is shown to be capable of monitoring and charting the dynamic frequency response of the cell to an electromagnetic field.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

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.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.     

Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-alpha on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-alpha, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately 3 days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, 300 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-alpha exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-alpha was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-alpha may regulate growth and functional activities of normal T cells.     

Capacity of tumor necrosis factor to bind and penetrate membranes is pH-dependent

Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4. A detailed examination of TNF-membrane interactions revealed that although TNF bound avidly to model membrane targets, no damage was generated under any condition tested. Binding of TNF, monitored with 125I-labeled as well as unlabeled protein, was enhanced at low pH. In the pH range tested (i.e., 4 to 8), target membrane permeability actually decreased in the presence of TNF. This membrane stabilization may be a consequence of TNF insertion into the target bilayer, a process we detected through use of an intramembranous photolabeling assay; interestingly, the efficiency of TNF insertion into membranes increased dramatically with decreasing pH. We conclude that native TNF does not cause pore formation directly and that its ability to induce cell lysis, as monitored by 51Cr release, is a consequence of some as yet obscure signaling event or intracellular activity. Parallel studies were carried out with diphtheria toxin, a protein with a more thoroughly characterized pH-dependent intoxification pathway. This toxin displayed acid-enhanced activities with both biologic and artificial targets.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

Differential incorporation of docosahexaenoic acid into distinct cholesterol-rich membrane raft domains

We investigated the influence of docosahexaenoic acid (DHA) on the fatty acid and protein compositions of two populations of membrane rafts present in Caco-2 cells. DHA (100 microM) had no significant influence on the fatty acid or protein compositions of tight junction-associated, Lubrol insoluble, membrane rafts. However, DHA did significantly alter the fatty acid and protein compositions of "archetypal" Triton X-100 insoluble membrane rafts. The DHA content of the raft lipids increased 25-fold and was accompanied by a redistribution of src and fyn out of the rafts. DHA also increased Caco-2 cell monolayer permeability producing a 95% drop in transepithelial electrical resistance and a 8.56-fold increase in the flux of dextran. In conclusion, the data demonstrate that DHA does not increase permeability through modifying the TJ-associated rafts. The data do, however, show that DHA is differentially incorporated into different classes of membrane rafts, which has significant implications to our understanding of how omega-3 PUFAs modulate plasma membrane organization and cell function.     

重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段,直接克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定后,再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间,构成重组质粒ｐＫＧ－ｈＦＡＳ,将此质粒导入大肠杆菌,经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达,用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白,经凝血酶酶切和二次亲合层析去除ＧＳＴ部分,得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体,经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡     

Active,soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U‐87 MG glioma cells and inhibits growth of the gram‐positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ～0.5–1 mg/L of E. coli culture. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1150–1157, 2013     

Overproduction of fungal ribotoxin α-sarcin in Escherichia coli: generation of an active immunotoxin

α-Sarcin is a ribonucleolytic protein secreted by the mold Aspergillus giganteus. DNA encoding α-sarcin was isolated from the host and cloned into T7 promoter based E. coli expression vectors. Using bacterial outer membrane protein A (OmpA) signal sequence, properly processed recombinant (re-) protein was secreted into the culture medium while in the absence of a signal sequence protein remained insoluble in the bacterial inclusion bodies. The re-α-sarcin was purified to homogeneity by simple chromatographic techniques both from the insoluble and soluble sources with respective yields of 40–50 μg/ml and 2–3 μg/ml. The re-ribotoxin was functionally as active as the native toxin and preserved its specificity. The re-α-sarcin was used in the construction of an active immunotoxin targeted at the human cancer cells overexpressing transferrin receptor (TFR).