A biologically active lectin of enteroaggregative Escherichia coli

The pathogenesis of enteroaggregative Escherichia coli, a major contributor to paediatric diarrhoea, is still not clearly understood. A complex carbohydrate specific lectin was identified from the culture supernatant of an enteroaggregative E. coli strain. The lectin was purified to 660-fold by a combination of sequential saturated ammonium sulphate precipitation and gel filtration chromatography in the FPLC system. The homogeneity of the purified lectin was established by analytical isoelectrofocusing [pI 6.75]. Hemagglutination of rabbit erythrocytes by the purified lectin was best inhibited by fetuin. The N-terminal sequence of the 41.7 kDa subunit showed homology to the outermembrane porins and the 23.4 kDa subunit showed homology to a hypothetical protein of Yersinia pestis and secreted Hcp protein. This protein could induce extensive morphological changes in HEp-2 cells and significant amount of fluid accumulation in rabbit ileal loop. GM1 showed maximum binding to the lectin among all other gangliosides. This purified protein showed cross-reactivity to the binding subunit of cholera toxin in western immunoblot. The presence of this toxin in some of the clinical isolates of enteroaggregative E. coli was also observed. The structural and functional characteristics of the toxin revealed that it is a novel virulence determinant of aggregative E. coli.     

High-level production of biologically active chemokines in Escherichia coli

The chemokines eotaxin-1 (CCL11) and eotaxin-2 (CCL24), belonging to the CC chemokines family, play key roles in the inflammatory response, allergic asthma and other diseases. When expressed in Escherichia coli, chemokines are prone to form inclusion bodies devoid of biological activity, and it is hard to refold them properly. Here an expression and purification protocol for high-level production of soluble and biologically active CCL11 and CCL24 in E. coli has been established. A final yield of 8.7 mg/l for CCL11 and 3.9 mg/l for CCL24 has been obtained and the purified proteins were characterized with SDS-PAGE, mass spectrometry and circular dichroism. High binding affinity of purified chemokines with CC chemokine receptor type 3 (CCR3) has been confirmed with surface plasmon resonance (SPR) and the K_D values are 3.7 × 10⁻⁷ M and 3.0 × 10⁻⁷ M, respectively, for CCL11 and CCL24. This report provides a straightforward strategy for the efficient production of soluble and biologically active chemokines in E. coli.     

Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies

Glutamyl-tRNA reductase catalyzes the initial step of tetrapyrrole biosynthesis in plants and prokaryotes. Recombinant Escherichia coli glutamyl-tRNA reductase was purified to apparent homogeneity from an overproducing E. coli strain by a two-step procedure yielding 5.6 mg of enzyme per gram of wet cells with a specific activity of 0.47 micromol min(-1)mg(-1). After recombinant production, denatured glutamyl-tRNA reductase from inclusion bodies was renatured by an on-column refolding procedure. Residual protein aggregates were removed using Superdex 200 gel-filtration chromatography. Solubility, specific activity, and long-term storage properties were improved compared to previous protocols. Obtained enzyme amounts of high purity now allow the research on the recognition mechanism of tRNAGlu and high-throughput inhibitor screening.     

Recombinant expression of biologically active rat leptin in Escherichia coli

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.     

Stopped flow method at subzero temperatures

A stopped-flow apparatus especially adapted for experiments with aqueous-organic solutions of enzymes at subzero temperatures is described.Performance data are given and discussed in a study of the reaction between ascorbate and 2,6-dichlorophenol-indophenol (DCPIP) in an “antifreeze” aqueous-organic mixture as a function of temperature.     

Formation of biologically active peptides

Many small biologicaly active peptides are derived from larger precursor forms which fulfil a variety of roles in the synthesis, segregation and intracellular migration of secretory products. Limited proteolysis may occur at several stages during this process, giving rise to products that are either degraded (e.g. the prepeptides) or discharged coordinately from their cells of origin during exocytosis (e.g. insulin and C-peptide). Molecular defects have recently been found to occur at cleavage sites in proinsulin as well as in other proproteins, and these point mutations may, in some instances, be responsible for familial metabolic disorders. The nature and cell specificity of the proteolytic enzymes involved in the conversion of the various precursor forms remains unresolved. Recent studies in our laboratory have led to the identification of precursors of glucagon and somatostatin in rat islets of Langerhans. Analysis of tryptic maps of these precursors has shown that a trypsin-like enzyme would be sufficient to cleave the C-terminally located somatostatin sequence from its precursor (relative molecular mass 12,500), but that both trypsin-like and carboxypeptidase B-like enzymes would be necessary to cleave the internal glucagon sequence from its prohormone (relative molecular mass 18,000). Molecular cloning techniques have provided valuable new approaches to analysing the structures of a variety of precursor forms, including those for insulin, gastrin, growth hormone, adrenocorticotropic hormone and the endorphins, and in the future will undoubtedly shed more light on the structures of their chromosomal genes, the mechanisms regulating their expression, and their evolutionary origins.     

Freeze-fixation at high subzero temperatures.

Previous attempts to determine the distribution of ice in frozen tissues at high sub-zero temperatures generally called for the further cooling of the tissues in question to facilitate freeze-drying, freeze-substitution, and freeze-fracture replication. Direct cryomicroscopic determinations, free from uncertainties stemming from changes in sample temperature could, it seemed, only be made in certain special cases. We have presented an isothermal “freeze-fixation” procedure designed to permit, instead, the postthaw retention of the freezing pattern and the conventional processing, afterward, of the thawed specimen. The method demands the exposure of the frozen tissues to fixative solutions incapable of dissolving ice. Frozen specimens are immersed in aqueous fixative solutions prepared in each instance (1) to freeze at a temperature equal to that at which fixation is to be conducted, (2) to contain quantities of finely divided ice sufficient to guarantee the maintenance of a constant water activity. Frozen frog and rat hearts and skeletal muscle tissues were exposed to formaldehyde, formaldehyde/ glutaraldehyde, and glutaraldehyde solutions at ?2, ?5, and ?10 °C, the temperatures being maintained in each case to ± 0.1 °C, or better. Tissues withdrawn at intervals were thawed, postfixed, dehydrated, embedded, and sectioned. The sections demonstrated the retention, after thawing, of structural features characteristic of the frozen state. The small hearts we exposed to formaldehyde were fixed throughout in 3 hr at ?2 ° and in 20 hr at ?5 °C. The action of osmium tetroxide was investigated. The method appears to be well-suited to numerous experimental applications.     

Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several alpha and beta human chemokines, suggesting that it is generally applicable to this class of proteins.     

Kinetics of water loss from cells at subzero centigrade temperatures

A mathematical model is developed for the calculation of the kinetics of water loss from cells at subzero centigrade temperatures. In this model it is assumed that the cell surface membrane is permeable to water only, the protoplasm is a nonideal solution, the cells are spherical, and during the cooling process the cell temperature is not uniform inside the cell. It is also assumed that because of water loss due to cooling process the cell volume and the cell surface area reduce and the reductions in surface area and volume of the cell are functions of the amount of water loss from the cell. Based on this model, and for different conditions, the fractions of supercooled intracellular water remaining in the cells at various temperatures are calculated.It is shown that for cooling cells at subzero centigrade temperatures. (1) the consideration of Clausius-Clapeyron equation for vapor pressures of water and ice, instead of the exact vapor pressure relations, may produce errors in the prediction of the amount of water loss from the cells at high cooling rates only, (2) the assumption of intact cells will produce considerable deviation in the prediction of water loss from the cells as compared to the more realistic assumption of shrinkable cells, (3) the nonideality of protoplasm solution is very effective on the prediction of the amount of water loss from the cells, and (4) the assumption of uniform-temperature cells during the cooling process may be erroneous only for cells with small fractions of water in their protoplasms.     

Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli.

The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.     

Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli

Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(121) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF(121) and VEGF(121)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF(121) and VEGF(121)/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF(121) (59.2mg/L) and 27-fold for VEGF(121)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(121) and VEGF(121)/rGel, respectively). The purified VEGF(121) and VEGF(121)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.     

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.     

The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli

Culture conditions affecting the formation of beta-galactosidase inclusion bodies in E. coli were examined. High temperature, early induction, high salt concentration and low aeration were all found to favour an increase of insoluble beta-galactosidase and the formation of visible inclusion bodies. The ratio of soluble to insoluble beta-galactosidase decreased during the course of cell growth. When assayed for beta-galactosidase activity, the inclusion bodies were enzymatically active with a specific activity of one third that of soluble beta-galactosidase. The activity remained associated with the inclusion bodies on washing with detergent and high ionic strength buffers. These results suggest that inclusion bodies can contain correctly folded protein.     

Production of biologically active Bacillus anthracis edema factor in Escherichia coli

Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.     

The expression of biologically active cholera toxin in Escherichia coli.

Chromosomal DNA from Vibrio cholerae El Tor strain 1621 was digested with Hind III and the products fractionated by centrifugation through a sucrose gradient. A 15kb fragment containing the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterised by digestion with different restriction endonucleases. Sequences homologous to the LT gene were identified by hybridisation and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.     

Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Isoelectric focusing and electrophoresis at subzero temperatures

Electrophoretic and isoelectric focusing separations have been achieved at ?20 to ?30°C, i.e., at temperatures considerably lower than previously reported by using as supporting media gels of acrylamide-methylacrylate copolymers and dimenthylsulfoxide-water mixtures. Hybrids of human and sickle cell hemoglobin and partially oxidized human carboxyhemoglobin have been separated in the temperature range ?20 to ?30°C, both by a discontinuous buffer gel electrophoresis and by isoelectric focusing.     

Expression of soluble, biologically active recombinant human endostatin in Escherichia coli

Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

High level synthesis of biologically active recombinant trichosanthin in Escherichia coli.

Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC50 was approximately 0.13 nM in an in vitro rabbit reticulocyte translational system and a dose of around 35 micrograms protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.