DEAE-Sepharose Fast Flow Research Articles

Isolation and Characteristic of Chondroitin Sulfate from Grass Carp Scales

Papain and alcalase were successively used to hydrolyze grass carp scales for preparation of crude polysaccharide. Chondroitin sulfate were further purified by DEAE-Sepharose Fast Flow and Sepharose 6 Fast Flow gel column chromatograph, and indentified by IR, NMR, element analysis and chemical analysis. Three polysaccharide fractions including chondroitin sulfate were fistly obtained from grass carp scales. The weight-average molecular weight of chondroitin sulfate was 46168 Da. The contents of various elements (C, H, N, S, O) in chondroitin sulfate were 28.89 %, 3.80 %, 3.36 %, 6.71 % and 47.16 %, respectively. The results of IR, NMR and chemical analysis exhibited that the sulfate group in chondroitin sulfate be O-linked in position 4 of N-acetyl-D-galactosamine.

Preparation and anticoagulant activity of a fucosylated polysaccharide sulfate from a sea cucumber Acaudina molpadioidea

Abstract A fucosylated polysaccharide sulfate, AMP-2, was purified by DEAE-Sepharose Fast Flow and Sephadex G-100 columns in successive steps from a special sea cucumber in southeastern China. HPLC and cellulose acetate membrane electrophoresis experiments confirmed AMP-2 was a homogenous carbohydrate with a relative molecular weight of ca. 2.4 × 10 4 Da, and methylation analysis indicated that polysaccharide was composed of 1-substituted-Gal p , 1,4-disubstituted-GalN p , 1,2-disubstituted-FucS p , 1,4,6-trisubstituted-Glc p in a molar ratio of ca. 0.5:2.0:1.0:3.0, together with a small amount of different substituted Man p . Sulfated derivative and carboxymethylated derivative were prepared using dry pyridine and chlorosulfonic acid, and chloroacetic acid, respectively. Anticoagulant activities in vitro investigation showed that sulfated derivative showed a stronger ability than native polysaccharide and carboxymethylated derivative, which might be caused by their different percentages and types of functional groups in their structures.

Purification and physicochemical properties of different polysaccharide fractions from the water extract of Boschniakia rossica and their effect on macrophages activation

Today more and more attentions had been attracted by many nutritionists and pharmacologists on polysaccharides from natural plants or animals due to their significant biological activities. In this research three polysaccharides (BRR-W1, BRR-WA1 and BRR-WA2) were isolated and purified from the water extract of Boschniakia rossica by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Chemical and physical characteristics of three polysaccharides were investigated by a combination of chemical and instrumental analysis methods. The assays of their effect on macrophages activation were also investigated in vitro, including phagocytosis of macrophages, detections for NO production and TNF-α secretion. The results indicated that the effect of polysaccharides on macrophages activation was influenced by their respective physicochemical properties.

Characterization and antioxidant activity of Ginkgo biloba exocarp polysaccharides

Ginkgo biloba exocarp polysaccharide (GBEP) was obtained by hot water extraction, the crude polysaccharide was deproteinized by Sevag method and fractionized by a DEAE Sepharose fast flow anion-exchange column. Five fragments were obtained, including neutral polysaccharide (GBEP-N) and four acidic polysaccharides (GBEP-A1, GBEP-A2, GBEP-A3 and GBEP-A4). GBEP-N and GBEP-A3 were further purified by Superdex 200 gel column chromatography. The resulted two fractions GBEP-NN, and GBEP-AA were characterized by FT-IR, and HPGFC (high pressure gel filtration chromatography). Monosaccharide composition was determined by RP-HPLC method of precolumn derivatization with 1-phenyl-3-5-pyrazolone. GBEP-NN was mainly composed of rhamnose, arabinose, mannose, glucose and galactose, while GBEP-AA was mainly made up of mannose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose. The crude GBEP exhibited certain antioxidant activity. At the concentration of 5mg/mL, the hydroxyl radical scavenging effect of GBEP was 90.52%, greater than 77.37% for the positive control ascorbic acid.

Purification and identification of one glucan from golden oyster mushroom (Pleurotus citrinopileatus (Fr.) Singer).

One water-soluble glucan (PCP-W1) was purified from the crude polysaccharide of Pleurotus citrinopileatus by chromatography on DEAE Sepharose Fast Flow and Sephadex G-200 column, and PCP-W1 (Mw=45kDa), was predominantly composed of Glc. Partial acid hydrolysis, Smith degradation-periodate oxidation, methylation analysis, Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS) analysis and nuclear magnetic resonance spectroscopy (NMR) experiments were conducted to elucidate its structure. The results indicated that PCP-W1 had a glucan backbone consisting of (1→6)-linked-β-d-glucopyranosyl residues, which were branched at O-3 position of the backbone with (1→3)-linked-β-d-glucopyranosyl and non-reducing end 1-β-d-glucopyranosyl residues. The repeating unit of the polysaccharide was established as.

High-level Expression of Mycobacterium tuberculosis Protein EspB in E. coli and Preparation of Novel Anti-EspB Monoclonal Antibodies

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis. The ESX-1 secreted protein EspB is cleaved close to its C-terminus during secretion and is necessary for inhibiting phagosome maturation. In this study, the EspB gene of M. tuberculosis H37Rv was cloned into the expression vector of pET21a(+) and was effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as a soluble form in cell lysate and was first purified by a column packed with Ni-NTA resin and then a column packed with DEAE-Sepharose Fast Flow matrix. Using the purified protein to immunize BALB/c mice, five monoclonal antibodies were produced. As shown by ELISA and immunoblotting, the five respective antibodies could recognize the EspB protein. These monoclonal antibodies will provide powerful reagents for further investigation of EspB protein functions.

Structural elucidation of a heteroglycan from the fruiting bodies of Agaricus blazei Murill

One water-soluble polysaccharide (ABP-W1) was purified from the fruiting bodies of Agaricus blazei by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Its molecular weight was about 3.9×102kDa as determined by high-performance size-exclusion chromatography (HPSEC). The structural feature of ABP-W1 was investigated by a combination of chemical and instrumental analysis, including partial hydrolysis with acid, periodate oxidation–Smith degradation, acetylation, methylation analysis and nuclear magnetic resonance spectroscopy (NMR 1H, 13C). The results revealed that ABP-W1 had a backbone consisting of (1→6)-linked-α-d-galactopyranosyl and (1→2,6)-linked-α-d-glucopyranosyl, which was branched with one single terminal (1→)-α-d-glucopyranosyl at the O-2 position of (1→2,6)-linked-α-d-glucopyranosyl along the main chain in the ratio of 1:1:1. The observation of the complex-formation between ABP-W1 and Congo Red indicated that ABP-W1 probably existed in a triple-strand helical conformation in water. Based on the data obtained, ABP-W1 was composed of a repeating unit with a structure as below: [Display omitted]

Antioxidant and immunoregulatory activity of different polysaccharide fractions from tuber of Ophiopogon japonicus

Four sulfated heteropolysaccharide fractions (OJP-1, OJP-2, OJP-3 and OJP-4) were isolated and purified from the tuber of Ophiopogon japonicus by DEAE-Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. OJP-1, the least sulfated product, was sulfated synthetically to OJP-1S. Their chemical–physical properties were determined by chemical methods, GC, FT-IR spectroscopy, and HPLC. The antioxidant and immunomodulation activities of these fractions were also investigated. The content of hexuronic acid and sulfate were decreased in the order OJP-4>OJP-3>OJP-2>OJP-1 and OJP-1S>OJP-4>OJP-3>OJP-2>OJP-1, respectively. In comparison with OJP-1, other polysaccharides showed stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydroxyl radical scavenging activity. Five polysaccharides also exhibited remarkable macrophage-activating capability by the promotion of phagocytic capacity, energy metabolism rate, NO and interleukin-1 production. It was significantly different between five polysaccharides (P<0.01) and decreased in the order OJP-1S>OJP-4>OJP-3>OJP-2>OJP-1. Taken together, our results suggested that hexuronic acid and sulfate were effective indicators of antioxidant and immunomodulation activity of polysaccharide.

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

A one-step purification process using flow-through mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min−1. Ft-IEC using Tris–HCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris–HCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.

Development and evaluation of flow through technique for diagnosis of cystic echinococcosis in sheep

A novel in vitro flow through technique was developed and evaluated for immunodiagnosis of cystic echinococcosis in sheep using hydatid specific non-cross reactive 8-kDa protein. The 8-kDa protein was prepared from hydatid cyst fluid by DEAE-Sepharose fast flow anion exchange chromatography. In this flow through technique, the 8-kDa antigen was coated on the nitrocellulose membrane of flow through device. Protein A colloidal gold was used as detector. The evaluation of the technique was performed by comparing 150 known positive hydatid serum and known negative serum collected from sheep. The test was shown to be high sensitivity and specificity that were closely correlated with those of EITB. Furthermore the immunofiltration-based assay is rapid (2min) and easy to perform with no requirement of special skill, reagent and instrumentation. This suggests the flow through technique is an acceptance alternative to be used in clinical laboratories lacking specialized equipments as well as large scale screening of cystic echinococcosis both in the field with animal and human populations.

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

Biocontrol Efficiency of Bacillus subtilis SL-13 and Characterization of an Antifungal Chitinase

The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato. The fresh and dry weight of tomato seedlings increased 42.86% and 18.75%, respectively. The control efficacies of the SL-13 to tomato Rhizoctonia rot were 20.65% and 35.23% in the greenhouse and field, respectively. The growth of the plant-pathogenic fungus Rhizoctonia solani was considerably inhibited in the presence of the strain SL-13 culture supernatant. The main antifungal protein was detected to be chitinase through vitro assay. The chitinase was purified with DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration for further characterization. The optimal pH and temperature for the chitinase activity were 7.0 and 50 (C, respectively. It was demonstrated that the enzyme was stable at pH 5-9 and 40-60 (C. 70% of the enzyme activity was retained when incubated at 121 (C and 0.11 MPa for 20 min, and the enzyme was not sensitive to protease K and ultraviolet radiation. Thus it is suitable for effective biological control in relatively unstable environment.

Purification and some properties of endoglucanase from Glyptotermes xiamenensis

AbstractAn endoglucanase (EGase) from Glyptotermes xiamenensis was purified by extraction with phosphate buffer solution and ammonium sulfate fractionation, and then chromatographed on DEAE Sepharose Fast Flow followed by Sephadex G‐100 columns. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE). Enzyme molecular weight was determined to be 44.8 kDa and the enzyme was a monomeric protein. The specific activity was determined to be 458.7 U/mg. The optimum pH and optimum temperature of the enzyme for the hydrolysis of carboxymethycellulose sodium (CMC‐Na) were investigated to be at pH 5.0 and at 40°C, respectively. The enzyme was stable over the pH range from 4.0 to 9.0 and at the temperature below 45°C. Kinetic behaviour of the enzyme in the hydrolysis of CMC‐Na followed Michaelis–Menten kinetics with constant (Km) of 7.2 mg/ml at pH 5.0 and 40°C. The activation energy was determined to be 49.3 KJ/mol. Chemical modification indicated that the residues of histidine, tryptophan and carboxylate were essential to the enzyme activity.

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn2+, Sn2+, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl b-D-xylopyranoside. The apparent Km value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3±0.236 and 60.2±6.788mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.

Purification and structural characterization of a new water-soluble neutral polysaccharide GLP-F1-1 from Ganoderma lucidum

A water-soluble neutral polysaccharide (GLP-F1-1) was isolated from the fruiting bodies of Ganoderma lucidum by DEAE Sepharose Fast Flow and Sephacryl S-500 High Resolution Chromatography. The neutral polysaccharide had an average molecular weight ( M w) of approximately 2.5 × 10 6 kDa. GC analysis showed that this polysaccharide was mainly composed of glucose and galactose in the molar ratio of 34:1. 1H and 13C NMR spectroscopy in combination with GC–MS technique indicated that the new polysaccharide had a backbone chain of 1,4-disubstituted-β-glucoseopyranose and 1,4,6-trisubstituted-β-glucoseopyranosyl, while the branched chains were mainly composed of 1,6-disubstituted-β-glucopyranosyl and 1,4-disubstituted-β-galactoseopyranosyl residues.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography-mass spectrometry (LC-MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS-PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60-70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.

Purification and Properties of Endoglucanase from a Sugar Cane Bagasse Hydrolyzing Strain, Aspergillus glaucus XC9

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 degrees C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 degrees C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 degrees C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis-Menten kinetics with constant K(m) of 5.0 mg/mL at pH 4.0 and 50 degrees C. The enzyme activity was stimulated by Fe(2+) and Mn(2+) but inhibited by Cd(2+), Pb(2+), and Cu(2+). The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

Physicochemical and Structural Properties of 8S and/or 11S Globulins from Mungbean [Vigna radiata(L.) Wilczek] with Various Polypeptide Constituents

Two kinds of globulins, 8S and 11S globulins, with various polypeptide constituents, were well fractionated from acid- and salt-extracted mungbean globulins using DEAE-Sepharose fast flow column chromatography. The physicochemical and conformational properties, including amino acid composition, surface charge and hydrophobicity, free sulfhydryl group (SH) and disulfide bond (SS) contents, protein solubility, thermal and emulsifying properties, as well as secondary and tertiary conformations, were evaluated. Remarkable differences in polypeptide composition, surface charge and hydrophobicity, SS contents, protein solubility, thermal and emulsifying properties, and secondary and tertiary conformations were observed between 8S and 11S globulins. The physicochemical and conformational properties of the vicilins also varied with the heterogeneity of their polypeptides, but to a relatively limited extent. The emulsifying ability of these globulins was distinctly dependent on their protein solubility (or net charge), surface hydrophobicity and polypeptide heterogeneity. The thermal properties were similar among various vicilins, but distinctly different between the vicilins and 11S globulins. The circular dichrosim spectral analyses revealed that there were no marked differences in secondary and tertiary conformations between various vicilins, but the secondary, tertiary and quaternary conformations of 11S globulins were much more unordered and flexible than the vicilins. These results suggested good relationships between the physicochemical properties and conformational features of these globulins from mungbean, which could be useful for the utilization of these proteins in the food industry, and providing a working direction of mungbean breeding or protein engineering to improve its physicochemical properties.

Antioxidant activities of different polysaccharide conjugates (CRPs) isolated from the fruiting bodies of Chroogomphis rutilus (Schaeff.: Fr.) O. K. Miller

Four water-soluble purified polysaccharides (CRPsA-1, CRPsA-2, CRPsB-1 and CRPsB-2) were obtained from the fruiting bodies of Chroogomphis rutilus by DEAE Sepharose Fast Flow column chromatography and Sepharose 6 Fast Flow column chromatography. Their chemical and physical characteristics were determined by chemical methods, gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). On the basis of hydroxyl radical assay, superoxide radical assay and Fe 2+-chelating ability assay, the antioxidant activities of CRPsA-1, CRPsA-2, CRPsB-1 and CRPsB-2 were investigated. Among these fractions, CRPsB-1 and CRPsB-2 containing glucuronic acid had the higher scavenging effects on hydroxyl radicals and superoxide radicals, and more potent Fe 2+-chelating activity than CRPsA-1and CRPsA-2 containing no glucuronic acid in a concentration-dependent manner.