High-yield two-step chromatographic procedure for purification of fatty acid-binding protein from human heart.

A high-yield procedure for the purification of cytoplasmic fatty acid-binding protein from human heart (H-FABP) is described. H-FABP was purified by gel permeation chromatography on a Sephacryl S-200 column followed by anion-exchange chromatography on a Sepharose Q fast-flow column at pH 7.0. At this pH H-FABP binds strongly to the column and can be selectively eluted with a salt gradient. The two-step procedure showed a high degree of reproducibility. On average 50 mg of H-FABP was obtained from 150 g of human heart tissue, which corresponds to a recovery of about 50%. Purity was confirmed by gel electrophoresis and isoelectric focusing. Binding of oleic acid to purified H-FABP, using the Lipidex 1000 assay, revealed a maximal binding of 0.75 +/- 0.01 mol fatty acid/mol protein and a dissociation constant of 0.19 +/- 0.01 microM.     

Surface-modified membranes as a matrix for protein purification.

Recently introduced membrane-based chromatographic supports for protein separation are available either with a coupled ligand, e.g., protein A, protein G or ion-exchange groups, or as activated matrices for coupling a desired ligand. The coupling conditions for protein A and immunoglobulin G to an epoxy-activated membrane were determined. The performance of the prepared affinity membranes was investigated using pure rabbit immunoglobulin G and protein A as a model system. For practical application monoclonal antibodies from cell culture supernatant were purified with a prepared protein A membrane and for comparison with a sulphonic acid ion exchange membrane.     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.     

Single-step method for purification of human transferrin from a by-product of chromatographic fractionation of plasma.

A rapid, simple and convenient method is described for the isolation, on a pilot scale, of pure and functional human transferrin from an unexploited by-product of chromatographic fractionation of plasma. In a single chromatographic step on DEAE-Spherodex, 97% pure transferrin was obtained in 75% yield. A virus inactivation treatment was included in the preparative process in order to guarantee the safety of the final product, which could be used in culture media.     

Monosized adsorbents for high-performance affinity chromatography. Application to the purification of calf intestinal alkaline phosphatase and human urine urokinase

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphatase and human urine urokinase from crude extracts. This study demonstrates that the combination of specifically designed affinity ligands with monosized support materials is a powerful approach to the resolution of proteins by high-performance affinity chromatography.     

Immobilized urokinase column as part of a specific detection system for plasminogen species separated by high-performance affinity chromatography

Immobilized urokinase was used as part of a post-column reactor for the specific detection of human plasminogen species which were fractionated using a high-performance affinity column. After on-line activation of each peak, plasmin activity was measured by mixing the eluate with a specific fluorogenic substrate and the product was detected by a fluorescence monitor. This detection system gave linear calibration graphs for both purified plasminogens (0.1-50 micrograms) and plasminogens contained in plasma (25-100 microliters). Relative standard deviations for the determination of plasminogens in plasma were 6.1-6.6% (n = 12), showing good reproducibility. The detection limit was as low as 0.1 micrograms of plasminogen. Immobilized urokinase was very stable and no appreciable decrease in activity was found after 100 cycles of operation. In combination with an immobilized benzamidine column, this system made it possible to separate and detect Glu-plasminogen and Lys-plasminogen contained in human plasma samples as small as 100 microliters without any pretreatment.     

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.     

Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.     

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.     

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.

Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.     

Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification.

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.     

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.     

Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent

A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N'-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10-100 microm size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 degrees C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts.     

A simple procedure for the chromatographic isolation and determination of prostaglandins from human seminal plasma

Summary A relatively simple procedure for the isolation and determination of the prostaglandins present in human seminal fluid is described. It involves preliminary chromatographic purification of these compounds from the major non-prostaglandin impurities followed by their total elution in one solvent (one-step elution). The prostaglandins thus obtained were almost free from other lipids and were further resolved into prostaglandin-groups and individual prostaglandins by repeated thin-layer chromatography. Data are also presented for prostaglandin contents of fresh semen samples from five individuals and results compared with those from the stored samples.

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Einfaches Verfahren zur chromatographischen Isolierung und Bestimmung von Prostaglandinen aus menschlichem Sperma

Zusammenfassung Das Verfahren umfaßt eine chromatographische Abtrennung der Verbindungen von den hauptsächlichsten Verunreinigungen und die Gesamtelution mit einem Lösungsmittel. Die von anderen Lipiden fast völlig freien Prostaglandine werden durch wiederholte Dünnschicht-Chromatographie in Gruppen und Einzelverbindungen getrennt. Werte werden angegeben über die Prostaglandingehalte von frischem im Vergleich zu gelagertem Sperma.

     

The study for isolation and purification of R-phycoerythrin from a red alga

An effective procedure for the rapid extraction and purification of the biliprotein R-phycoerythrin from a red alga,Ceramium isogonum, was developed. The purified R-phycoerythrin ofC. isogonum consisted of three components with mol wt 180,000 (6β subunits), 70,000 (6α subunits), and 30,400 (γ subunit), respectively. The phycoerythrin is suitable for use as a natural food coloring and can also be used as a fluorescent label.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.     

Model-based design of a pilot-scale simulated moving bed for purification of citric acid from fermentation broth

One of the conventional processes used for the recovery of citric acid from its fermentation broth is environmentally harmful and cost intensive. In this work an innovative benign process, which comprises simulated moving bed (SMB) technology and use of a tailor-made tertiary poly(4-vinylpyridine) (PVP) resin as a stationary phase is proposed. This paper focuses on a model-based design of the operation conditions for an existing pilot-scale SMB plant. The SMB unit is modeled on the basis of experimentally determined hydrodynamics, thermodynamics and mass transfer characteristics in a single chromatographic column. Three mathematical models are applied and validated for the prediction of the experimentally attained breakthrough and elution profiles of citric acid and the main impurity component (glucose). The transport dispersive model was selected for the SMB simulation and design studies, since it gives a satisfactory prediction of the elution profiles within acceptable computational time. The equivalent true moving bed (TMB) and SMB models give a good prediction of the experimentally attained SMB separation performances, obtained with a real clarified and concentrated fermentation broth as a feed mixture. The SMB separation requirements are set to at least 99.8% citric acid purity and 90% citric acid recovery in the extract stream. The complete regeneration in sections 1 and 4 is unnecessary. Therefore the net flow rates in all four SMB sections have been considered in the unit design. The influences of the operating conditions (the flow rate in each section, switching time and unit configuration) on the SMB performances were investigated systematically. The resulting SMB design provides 99.8% citric acid purity and 97.2% citric acid recovery in the extract. In addition the citric acid concentration in the extract is a half of its concentration in the pretreated fermentation broth (feed).     

Improved affinity chromatographic purification of D-mannose-N-acetyl-D-glucosamine-specific lectin from the bark of Sophora japonica eliminating the loss by sugar specific self-aggregation.

A novel D-mannose-N-acetyl-D-glucosamine-specific lectin of Sophora japonica bark, B-SJA-II, which showed self-aggregation based on sugar specificity, was purified by affinity chromatography on maltamyl-Sepharose subsequent to chromatographic separation on lactamyl-Sepharose to remove a major D-galactose-N-acetyl-D-galactosamine specific lectin, B-SJA-I. However, the yield of this method was low as a result of the sugar-specific precipitation and binding to other glycoproteins. A modified method was developed to circumvent this problem. All the purification procedures, except for the final chromatographic separation, were carried out in the presence of the haptenic sugar and the sugar-specific adsorption of B-SJA-II onto the adsorbent was carried out in a dialysis bag by gradually removing the sugar. This method gave a yield eight times higher than the original method.     

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver.

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30-70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant (KM) and catalytic constant (Kcat)] and the ratio Kcat/KM against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 micrograms of cathepsin B and 50 micrograms each of cathepsins L and H are obtained in a single run.