Sepharose CL Research Articles

Isolation of CA 125 from Breast Tumor Tissue Using Gel Chromatography

Gel filtration chromatography technique was used (Sepharose CL - 68) of CA 125 from malignant breast tumor homogenate and the isolation of (CA 125 I 1251 - anti CA 125 antibody), complex was prepared from crude CA 125 , isolated CA 125 and standard human CA 125. The results revealed the presence of two forms of CA 125 (Agl & AgII) and found to be 517 Kda and 38 Kda respectively; The isolation of CA 125 shov,:ed a purification fold of 17.5 Agl and 15 for AgII.

A new thermostable rhizopuspepsin: Purification and biochemical characterisation

The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25−65 % saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 °C, respectively. The enzyme was unusually thermostable, as it retained 50 % of its activity beyond 85 °C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63 %) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with “experimental evidence at protein-level”.

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA-Sepharose CL 4B supports, for the use in bioconversion of residual colorants.

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2±1.3 vs. 12.1protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

Evaluating the ion exchange chromatography for matrix-assisted PEGylation and purification of consensus interferon.

Scientists have implemented protein-PEGylation technology for boosting-up the pharmacokinetics and stability of recombinant therapeutic proteins. In the present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. DEAE Sepharose CL 6B, DEAE Fracto gel, and Macro cap Q ion exchange chromatography medium were compared for on column PEGylation and purification of cIFN. A MSC-PEG of 12.0 KDa was selected. cIFN was bound to ion exchange medium, and PEG solution was passed through resin for 180 Min, and protein was eluted by sodium chloride linear gradient. Yield and purity for mono-PEGylated cIFN with Macro cap Q matrix was 75% and 99%, respectively, whereas for DEAE Sepharose was 45% and 60%. DEAE Fracto gelTM purity was 85% with 50% yield of mono-PEGylated cIFN. Further investigation of in vitro biological activities demonstrated that about 30% antiviral activity was reduced as compared to unmodified cIFN. However, thermal stability was significantly improved. The present study proved that matrix-assisted PEGylation can improve the yield and purity of mono-PEGylated product, and Macro Cap resin provided the highest yield of a homogeneous product. In present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. Matrix-assisted PEGylation increases the yield of mono-PEGylated product and further Macro CapTM produced highest yield and purity of PEGylated cIFN.

Chromatography of Hedysarum extracts on modified Sepharose CL sorbents

Aim. To study the chromatographic efficiency of modified Sepharose CL-4B sorbents for separation, purification, and preparation of major components from Hedysarum theinum Krasnob. and Hedysarum neglectum Ledeb. root extracts.Materials and Methods. Sorption capacity of the native and modified sepharose was evaluated in both isocratic and gradient elution modes. 4-pyridinecarboxylic acid derivative [(4-hydroxy-3-methoxyphenyl)methylene]hydrazide (ftivazide) was used as a sorbent modifier ligand. We studied chromatographic properties of Sepharose CL-4B, Sepharose CL-6B, and azoepoxyadsorbent Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide, a chemically modified analogue of Sepharose CL-4B.Results. Sepharose CL-4B and Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide were found efficient carriers for liquid column chromatography of water-ethanol Hedysarum extracts. Complete elution of bioactive substances has been achieved exclusively by using sodium tetraborate as an eluent. We were able to identify flavonoids and tannins of different condensation in chromatographic fractions.Conclusion. Comparative chromatography of bioactive substances from Hedysarum root extracts is more efficient if employing Sepharose CL-4B instead of Sepharose CL-6B as a sorbent. Sepharose CL-4B-DEP-p-nitrobenzohydrazide-ftivazide demonstrates excellent separation capacity for tannins and flavonoids.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Purification, characterization, and bioactivities of a polysaccharide from mycelial fermentation of Bjerkandera fumosa

In this work, a novel polysaccharide (named DBFM3) was isolated from mycelia of Bjerkandera fumosa by DEAE-32 and Sepharose CL–6B column chromatography. High-performance gel permeation chromatography showed that DBFM3 was homogeneous, with an average molecular weight of 1.8×105Da. Structural characteristics of the purified fraction were investigated by high-performance liquid chromatography (HPLC), FT-IR, and NMR. HPLC analysis indicated that DBFM3 was composed of mannose, galacturonic acid, and galactose at molar ratios of 1:18.16:0.702. Spectral analysis suggested that DBFM3 had (1→6), (1→3,6), (1→3) linkages and pyranose conformation. Antioxidant assay in vitro showed that DBFM3 exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activity, and has protective effect against DNA damage and damage to SH-SY5Y cells induced by H2O2. Immunological tests indicated that DBFM3 significantly increased lymphocyte proliferation in vitro. Furthermore, DBFM3 increased the proliferation of lymphocytes in the presence of concanavalin A or lipopolysaccharide as mitogen.

Preparation of an immunoaffinity chromatography column for chloramphenicol binding

Anti-chloramphenicol rabbit antibody was covalently bound to Sepharose CL 4B-CNBr gel at 5, 10 and 15 mg antibody per mL gel and was fed into columns to prepare three respective immunoaffinity chromatography columns for chloramphenicol (CAP) binding. Examination of CAP-binding efficiency of these columns being fed with 0.5, 1 and 10 ng CAP showed that the column with 10 mg antibody per ml gel (IAC-CAP-10 column) could bind more than 90 % of the CAP amount having been fed into the column at all three examined CAP amounts. This IAC-CAP-10 column showed a coefficient of variants (CV) between columns upon CAP binding of 5.18 % for 1 ng fed CAP and 1.98 % for 10 ng, respectively, a loading capacity of 153,37 ± 10,32 ng CAP, stable CAP-binding performance during at least 2 years of preservation at 4 °C. The coupling of this IAC-CAP-10 column with liquid chromatography - tandem mass spectrometry LC MS/MS could make CAP analysis become possible with a limit of detection LOD of 0.033 ng and a limit of quantification LOQ of 0.101 ng. The column could also bind to the other two phenicol antibiotics: florfenicol and thiamphenicol with a binding efficiency of 90.73 % for florfenicol and 84.29 % for thiamphenicol.

Purification, partial characterization and bioactivity of sulfated polysaccharides from Grateloupia livida

Purification, preliminary characterization and bioactivity of polysaccharides from Grateloupia livida (GL) were investigated. Three water-soluble sulfated polysaccharide fractions (GLP-1, GLP-2 and GLP-3) were isolated and purified from the edible and medicinal red seaweed, Grateloupia livida (Harv.) Yamada by DEAE Sepharose CL–6B and Sephadex G-100 column chromatography, and chemical characterization was performed by HPGPC, GC–MS, FT-IR and SEM. In addition, anticoagulant activities were determined by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using normal human plasma in vitro. The antioxidant activities against DPPH and ABTS+ radicals were evaluated and compared. The molecular weights of GLP-1, GLP-2 and GLP-3 were 39.5, 60.4 and 3.36kDa, respectively. Monosaccharide analysis revealed that three polysaccharide fractions were homopolysaccharides and comprised of galactose only. Anticoagulant assays indicated that crude GLP, and purified GLP-1 and GLP-2 effectively prolonged APTT and TT, but not PT. All polysaccharide fractions exhibited significant in vitro antioxidant activities in a dose-dependent manner. GLP-2 showed consistently better anticoagulant and antioxidant activities compared with GLP, GLP-1 and GLP-3. These results demonstrate that sulfated polysaccharides isolated from Grateloupia livida can serve as readily available alternative natural sources of anticoagulant and antioxidant agents.

Assessment of the cytotoxic and genotoxic effects of TiO2 nanoparticles in A549 cells

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system.

Chemical composition and thermal properties of Chilean Araucaria araucana starch

Starch isolated from Chilean Araucaria araucana seed was characterized as a follow‐up on a previous study. The apparent amylose (AMM) content determined by potentiometric titration was 28%. The chain length distribution profile of amylopectin (AP) showed two peaks at polymerization degrees (DP) of 12 and 43–44. Around 51% of unit chains had DP in the range 13–24. The average DP of AP branch chains was 19.6%. The molecular weight distribution of A. araucana starch analyzed by Sepharose CL 2B is also reported. The ratio of blue value to total carbohydrates was 0.30 for the AP peak, which is characteristic of a low proportion of long lateral chains. The gelatinization temperature and enthalpy determined by DSC were 65.6°C and 14.5 J/g, respectively.

Low-cost purification of nisin from milk whey to a highly active product

Nisin is a natural peptide used as a preservative in a variety of food products, in which it inhibits mainly Gram-positive bacterial growth, including multidrug-resistant pathogens. However, its application range depends on the cost-effective production and purification of this molecule. Our group has previously produced nisin by Lactococcus lactis cultivation in milk whey, which is an industrial residue from dairy production. To our knowledge, no report used milk whey as a culture medium, although several investigators have purified nisin using different techniques. We thus aimed to establish a low-cost purification of nisin obtained by this process. Samples were diluted in ammonium sulphate, applied onto HIC columns (butyl sepharose CL 4B matrix), and eluted with Milli-Q water or PBS. Elution fractions were monitored for protein content and nisin antibacterial activity. Water elution resulted in purification factor values (270, commercial nisin; 775, nisin produced in-house) higher than those obtained with PBS elution. We concluded that purification of nisin does not require precipitation with ammonium sulphate, therefore allowing step/cost reduction. Moreover, purification from milk whey using HIC provides nisin with high activity and low salt content, which can further be applied to a variety of areas.

A thermostable exo-β-fructosidase immobilised through rational design

Thermotoga maritima exo-β-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl–Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04M) in a batch stirred tank reactor at 60°C was achieved with a productivity of 22.2g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5days at 60°C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli

An efficient affinity-purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4-Aminopyrrole-2-carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C8 column and reducing SDS-PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS-PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ~46 kDa. The desorption constant Kd and theoretical maximum absorption Qmax were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.

Role of Staphylococcus aureus cell wall protein A in the pathogenesis of the bacteria

This study dealt with 20 diagnosed isolates of Staphylococcus aureus. The haemagglutination test was used to investigate the presence of protein (A) in all S.aureus isolates. One isolate has been chosen as an optimal isolate to accomplish this study, because of its high content of protein (A). Crude protein (A) was extracted by two steps. The first is "Suspending in sodium azide"; the second was precipitation by ammonium sulphate. In order to get a purified protein (A) extract from crude protein A, purification was accomplished by two steps: Ion – exchange chromatography by DEAE – cellulose column was used in the first step, while Gel – filtration chromatography by Sepharose CL – 6B column was used in the second step. Double immunodiffusion in gel was used to investigate the preservation of the crude and purified protein (A) with its full function. Bradford method was used to estimate protein (A) extracts concentration (Crude, Partial purified, Purified). White mice (Balb C) were used to investigate the pathogenic effects of purified protein (A) .The injected mice with purified protein (A) showed general weakness which was very clear after the sixth injection . There were no phenotypic dermal change in the injection area and no dead mice as well. After killing and dissecting of all the mice, the organs of heart, spleen, liver, kidney, intestine, stomach and lung were eradicated. There were a great enlargement in spleen and a little in liver in all injected mice in comparison with the control mice. No phenotypic changes appeared in heart, intestine and stomach, but black spots were shown on the outer surface of kidney and liver tissue which were presented in all the injected mice. The most important results in this study are the great size of pathogenic effects in lung. The tissue seemed faint black with which was obvious in seven mice. The lung of the three others (some parts) showed during anatomy covered by blood. The histological check for heart, intestine and stomach showed no pathogenic effects resulted from protein (A) injection. In the spleen, there was a huge expanding in the white pulp region, with blood congestion while the spreading of megacaryocytes has increased. The liver showed mild degenerative change in liver cells with monocytes infiltration especially in the entrance region which appeared expanded. Also there was an increase in kupffer cells in all liver tissue. Kidney showed increase in mesengial cells which are considered the forming units of glomeruli. The histological check of lung included bloody congestions, oedema, inflammatory cells infiltration and alveolar spaces expanding forming ''Emphysema '' The lung was the most effected organ that showed tissue injury among other all organs.

Isolation, Purification and Characterisation of Low Molecular Weight Xylanase from Bacillus pumilus SSP-34

Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried out at 50 °C. K (m) and V (max) values, determined with oats spelts xylan were 6.5 mg ml⁻¹ and 1,233 μmol min⁻¹ mg⁻¹ protein, respectively, and the specific activity was 1,723 U mg⁻¹.

Expression, purification and partial characterization of a xanthine oxidase (XOD) in Arthrobacter sp.

A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ∼20mg crude protein from disrupted cells was subjected to the antibody medium, and ∼1.45mg protein was detected in unbinding fractions with ∼92.0% of activity. The extracted xanthine oxidase was ∼85% pure with native-PAGE analysis, and ∼90% pure with SDS-PAGE analysis, the yield of protein was ∼7.4%. The specific activity of the enzyme was 36.0U/mg. The native enzyme should be a dimer (∼280kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50°C. Furthermore, EDTA revealed almost no influences on the activity.

Study of specific interaction between nucleotides and dye support by nuclear magnetic resonance

The binding between four matrices (beaded cellulose, cellulose acetate, cellulose triacetate and Sepharose CL-6B) and beaded cellulose derivatized with a thiacarbocyanine dye with 5'-mononucleotides is investigated by Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) technique. This procedure intends to identify unspecific interactions between 5'-mononucleotides and matrices commonly used in affinity chromatography systems and also clarify the contribution of a thiacarbocyanine dye immobilized onto cellulose beads in a biorecognition process. The differences between non-derivatized and derivatized beaded cellulose evidence the contribution of thiacarbocyanine dye in the observed interaction. STD-NMR experiments show that Sepharose CL- 6B interact less with the 5'-mononucleotides comparatively with beaded cellulose. Indeed, beaded cellulose shows nonspecific interactions with almost all 5'-mononucleotides that compromises the specificity of the interaction between the thiacarbocyanine dye immobilized with the 5'-mononucleotides. The cellulose matrices where the hydroxyl groups are replaced by acetate and triacetate groups do not exhibit binding response to the 5'- mononucleotides, whereas the thiacarbocyanine dye contribution is evidenced by the reinforcement of the interactions with the sugar moiety of 5'-GMP and 5'-UMP and with base of 5'-AMP, 5'-CMP and 5'-TMP. This screening of the nucleotide atoms involved in the binding to the supports can be very useful in chromatography evaluations in which dye-affinity chromatography supports may be used, such as purification of nucleic acids.

Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25°C and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25°C and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.