The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Bo Mattiasson - One of the best experts on this subject based on the ideXlab platform.
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Smart Polymers and Protein Purification
Polymer-Plastics Technology and Engineering, 1998Co-Authors: Bo Mattiasson, M. B. Dainyak, I. Yu. GalaevAbstract:Abstract Smart polymers undergo fast and reversible changes in microstructure triggered by small changes of medium property (pH, temperature, ionic strength). These properties of smart polymers were exploited for the development of new protein purification techniques: Affinity precipitation, Affinity partitioning, and temperature-induced elution in Dye-Affinity Chromatography. Different smart polymers were used for Affinity precipitation: Eudragit S 100 (a copolymer of methacrylic acid and methyl methacrylate which precipitates on decreasing pH); stoichiomet-ric polyelectrolyte complexes of poly(methacrylate) and poly(N-ethyl-4-vinylpyridium bromide); poly(N-vinyl caprolactam), and poly(N-iso-propylacrylamide) (both precipitate on heating aqueous solutions to 35–40°C or increase in ionic strength). Recombinant thermostable lac-tate dehydrogenase from a thermophile Bacillus stear other mophilus was purified by Affinity partitioning in an aqueous two-phase polymer system formed by dextran and a copolymer of...
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Polymer-shielded Dye-Affinity Chromatography.
Journal of molecular recognition : JMR, 1996Co-Authors: Bo Mattiasson, Igor Yu. Galaev, Nandita GargAbstract:Polymer-shielded Dye-Affinity Chromatography is a form of Chromatography in which the Dye matrix forms complexes with a nonionic, water-soluble polymer such as poly(vinylpyrrolidone) or poly(vinyl alcohol), prior to the column Chromatography of a crude protein extract, the idea being that polymer shielding of the Dye will prevent nonspecific interactions between the target protein and the Dye. The concept of polymer shielding and a strategy for the rational selection of polymers suitable as shielding agents are presented.
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Dye-Affinity TECHNIQUES FOR BIOPROCESSING : RECENT DEVELOPMENTS
Journal of molecular recognition : JMR, 1996Co-Authors: Nandita Garg, Igor Yu. Galaev, Bo MattiassonAbstract:Textile or triazine Dyes play an important role as Affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in Dye-Affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-Affinity Chromatography has become a routine step in protein purification. New Dyes have been developed and used successfully in both traditional chromatographic mode and new modes like Affinity precipitation, polymer aqueous two-phase partitioning or expanded bed Chromatography. The specificity of Dye techniques has been increased by both purposeful designing of new Dyes and decreasing non-specific protein–Dye interactions with polymer shielding. One can envisage further development and ramification of Dye-Affinity techniuqes in protein purification.
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Polymer displacement in Dye-Affinity Chromatography
Journal of Chromatography A, 1995Co-Authors: Igor Yu. Galaev, Pär Arvidsson, Bo MattiassonAbstract:Displacement of lactate dehydrogenase from Dye-Affinity matrices with poly(ethyleneimine) (PEI) was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional non-specific elution while the purification factors were unchanged. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the Dye-ligands. Complete elution of bound protein is a characteristic feature of such a mechanism. Hence displacement with PEI may be a promising strategy for eluting proteins with reported low recoveries in Dye-Affinity Chromatography protocols.
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Interaction of Cibacron blue with polymers: implications for polymer-shielded Dye-Affinity Chromatography of phosphofructokinase from baker's yeast.
Journal of chromatography. A, 1994Co-Authors: Igor Yu. Galaev, Nandita Garg, Bo MattiassonAbstract:Abstract Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the Dye. The difference spectra for the PVP—Dye complex was typical of “electrostatic interaction spectra” at low ionic strength and typical of “hydrophobic interaction spectra” in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One Dye molecule was bound by a PVP segment with a molecular mass of 1000–1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One Dye molecule was bound by a polymer segment with a molecular mass of about 10 000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded Dye-Affinity Chromatography. This concept was successfully applied to the Chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.
Igor Yu. Galaev - One of the best experts on this subject based on the ideXlab platform.
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poly acrylamide allyl glycidyl ether cryogel as a novel stationary phase in Dye Affinity Chromatography
Journal of Applied Polymer Science, 2007Co-Authors: Nazan Demiryas, Igor Yu. Galaev, Nalan Tuzmen, Erhan Piskin, Adil DenizliAbstract:Poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] cryogel was prepared by bulk polymerization which proceeds in an aqueous solution of monomers frozen inside a glass column (cryo-polymerization). After thawing, the monolithic cryogel contains a continuous polymeric matrix having interconnected pores of 10-100 mu m size. Cibacron Blue F3GA was immobilized by covalent binding onto poly(AAm-AGE) cryogel via epoxy groups. Poly(AAm-AGE) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(AAm-AGE) monolithic cryogel was 6.84 g H2O/9 cryogel. Poly(AAm-AGE) cryogel containing 68.9 mu mol Cibacron Blue F3GA/g was used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.2 mg/g). The maximum amount of HSA adsorption from aqueous solution in acetate buffer was 27 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 74.2 mg/g). Desorption of HSA with a purity of 92% from Cibacron Blue F3GA attached poly(AAm-AGE) cryogel was achieved using 0.110 Tris/HCl buffer containing 0.5M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with poly(AAm-AGE) cryogel without significant loss in the adsorption capacity. (Less)
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Polymer-shielded Dye-Affinity Chromatography.
Journal of molecular recognition : JMR, 1996Co-Authors: Bo Mattiasson, Igor Yu. Galaev, Nandita GargAbstract:Polymer-shielded Dye-Affinity Chromatography is a form of Chromatography in which the Dye matrix forms complexes with a nonionic, water-soluble polymer such as poly(vinylpyrrolidone) or poly(vinyl alcohol), prior to the column Chromatography of a crude protein extract, the idea being that polymer shielding of the Dye will prevent nonspecific interactions between the target protein and the Dye. The concept of polymer shielding and a strategy for the rational selection of polymers suitable as shielding agents are presented.
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Dye-Affinity TECHNIQUES FOR BIOPROCESSING : RECENT DEVELOPMENTS
Journal of molecular recognition : JMR, 1996Co-Authors: Nandita Garg, Igor Yu. Galaev, Bo MattiassonAbstract:Textile or triazine Dyes play an important role as Affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in Dye-Affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-Affinity Chromatography has become a routine step in protein purification. New Dyes have been developed and used successfully in both traditional chromatographic mode and new modes like Affinity precipitation, polymer aqueous two-phase partitioning or expanded bed Chromatography. The specificity of Dye techniques has been increased by both purposeful designing of new Dyes and decreasing non-specific protein–Dye interactions with polymer shielding. One can envisage further development and ramification of Dye-Affinity techniuqes in protein purification.
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Polymer displacement in Dye-Affinity Chromatography
Journal of Chromatography A, 1995Co-Authors: Igor Yu. Galaev, Pär Arvidsson, Bo MattiassonAbstract:Displacement of lactate dehydrogenase from Dye-Affinity matrices with poly(ethyleneimine) (PEI) was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional non-specific elution while the purification factors were unchanged. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the Dye-ligands. Complete elution of bound protein is a characteristic feature of such a mechanism. Hence displacement with PEI may be a promising strategy for eluting proteins with reported low recoveries in Dye-Affinity Chromatography protocols.
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Interaction of Cibacron blue with polymers: implications for polymer-shielded Dye-Affinity Chromatography of phosphofructokinase from baker's yeast.
Journal of chromatography. A, 1994Co-Authors: Igor Yu. Galaev, Nandita Garg, Bo MattiassonAbstract:Abstract Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the Dye. The difference spectra for the PVP—Dye complex was typical of “electrostatic interaction spectra” at low ionic strength and typical of “hydrophobic interaction spectra” in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One Dye molecule was bound by a PVP segment with a molecular mass of 1000–1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One Dye molecule was bound by a polymer segment with a molecular mass of about 10 000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded Dye-Affinity Chromatography. This concept was successfully applied to the Chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.
Nandita Garg - One of the best experts on this subject based on the ideXlab platform.
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Polymer-shielded Dye-Affinity Chromatography.
Journal of molecular recognition : JMR, 1996Co-Authors: Bo Mattiasson, Igor Yu. Galaev, Nandita GargAbstract:Polymer-shielded Dye-Affinity Chromatography is a form of Chromatography in which the Dye matrix forms complexes with a nonionic, water-soluble polymer such as poly(vinylpyrrolidone) or poly(vinyl alcohol), prior to the column Chromatography of a crude protein extract, the idea being that polymer shielding of the Dye will prevent nonspecific interactions between the target protein and the Dye. The concept of polymer shielding and a strategy for the rational selection of polymers suitable as shielding agents are presented.
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Dye-Affinity TECHNIQUES FOR BIOPROCESSING : RECENT DEVELOPMENTS
Journal of molecular recognition : JMR, 1996Co-Authors: Nandita Garg, Igor Yu. Galaev, Bo MattiassonAbstract:Textile or triazine Dyes play an important role as Affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in Dye-Affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-Affinity Chromatography has become a routine step in protein purification. New Dyes have been developed and used successfully in both traditional chromatographic mode and new modes like Affinity precipitation, polymer aqueous two-phase partitioning or expanded bed Chromatography. The specificity of Dye techniques has been increased by both purposeful designing of new Dyes and decreasing non-specific protein–Dye interactions with polymer shielding. One can envisage further development and ramification of Dye-Affinity techniuqes in protein purification.
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Interaction of Cibacron blue with polymers: implications for polymer-shielded Dye-Affinity Chromatography of phosphofructokinase from baker's yeast.
Journal of chromatography. A, 1994Co-Authors: Igor Yu. Galaev, Nandita Garg, Bo MattiassonAbstract:Abstract Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the Dye. The difference spectra for the PVP—Dye complex was typical of “electrostatic interaction spectra” at low ionic strength and typical of “hydrophobic interaction spectra” in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One Dye molecule was bound by a PVP segment with a molecular mass of 1000–1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One Dye molecule was bound by a polymer segment with a molecular mass of about 10 000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded Dye-Affinity Chromatography. This concept was successfully applied to the Chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.
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Effect of poly(vinyl alcohol) treatment of the Dyematrix on the Chromatography of pyruvate kinase
Biotechnology Techniques, 1994Co-Authors: Nandita Garg, Igor Yu. Galev, Bo MattiassonAbstract:The utility of poly(vinyl alcohol) as a shielding polymer in Dye-Affinity Chromatography was studied. Difference spectroscopy was used to estimate the strength of the polymer-Dye complex. The target enzyme, pyruvate kinase (E.C. 2.7.1.40) from porcine muscle was purified on a Red HE3B-Sepharose column with 95% recovery by salt elution and 85% recovery with specific eluent.
Federico Javier Wolman - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous purification and immobilization of soybean hull peroxidase with a Dye attached to chitosan mini-spheres
Biocatalysis and Biotransformation, 2017Co-Authors: Lautaro Fidel Bracco, Federico Javier Wolman, Gustavo Levin, Agustin A. Navarro Del Cañizo, María Victoria Miranda, Osvaldo CasconeAbstract:Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by Dye Affinity Chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditi...
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Lactoperoxidase purification from whey by using Dye Affinity Chromatography
Food and Bioproducts Processing, 2017Co-Authors: Nicolás Urtasun, María Fernanda Baieli, Daniela Belén Hirsch, María C. Martínez-ceron, Osvaldo Cascone, Federico Javier WolmanAbstract:Abstract Bovine lactoperoxidase is a glycoprotein present in milk, whey and colostrum, which might be used in dairy, cosmetic, pharmaceutical, veterinary and agricultural applications due to its broad antimicrobial activity. Here, we describe a novel process for bovine lactoperoxidase purification by using Dye Affinity Chromatography. Eighteen triazine Dyes were immobilized on Sepharose 6B and screened for their performance as possible ligands. Five of the Dye-Sepharose matrices showed over 90% adsorption of bovine lactoperoxidase directly from whey without any pretreatment using the batch mode, and were thus selected for further adsorption and elution studies. The highest elution degree was obtained using 20 mM acetate buffer, pH 5.0, 2 M NaCl, as the eluent for all the matrices. Whey processed using the Reactive Red 4-Sepharose matrix in batch mode showed the highest bovine lactoperoxidase purification yield (86.5 ± 3.8%), purification factor (46.1 ± 1.1), and a relative purity higher than 80% according to SDS-PAGE gel densitometry. Whey processed using packed-bed column mode showed lower yields and additional whey pretreatments were needed for dynamic processing. The interaction between bovine lactoperoxidase and Reactive Red 4-Sepharose matrix was characterized using Langmuir isotherm model. The K d value was 0.21 ± 0.03 mg/mL and the Q max was 32.21 ± 1.24 mg/g. The results presented here suggest the potential application of the Reactive Red 4-Sepharose matrix to one-step purification of bovine lactoperoxidase from whey.
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Isolation of lactoferrin from whey by Dye-Affinity Chromatography with Yellow HE-4R attached to chitosan mini-spheres
International Dairy Journal, 2014Co-Authors: María Fernanda Baieli, Nicolás Urtasun, Osvaldo Cascone, María Victoria Miranda, Federico Javier WolmanAbstract:Abstract Novel integrated chromatographic methods need to be developed for specific, low-cost protein purification from raw materials. Here, a process for bovine lactoferrin (Lf) isolation from sweet whey was developed using cross-linked chitosan mini-spheres with immobilised Yellow HE-4R Dye as a low-cost ligand. The maximum adsorption capacity was between 51.14 and 58.28 mg Lf g −1 matrix. In addition, the mini-spheres adsorbed around 95% of the Lf present in the sweet whey and eluted more than 80% of the adsorbed Lf. A yield of 77% with purity greater than 90% was achieved in only one purification step. The purification process was efficient for three consecutive cycles without regeneration steps.
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Triazinic Dye ligand selection by surface plasmon resonance for recombinant lactoferricin purification
Process Biochemistry, 2013Co-Authors: Nicolás Urtasun, María Fernanda Baieli, Osvaldo Cascone, Federico Javier Wolman, Pablo Nicolas Romasanta, Marisa M. Fernández, Emilio L. Malchiodi, María Victoria MirandaAbstract:Abstract Bovine lactoferricin (Lfcin B) belongs to the antimicrobial peptide family, which is the first line of defense against pathogens in many organisms. Lfcin B has important applications due to its antiviral, antifungal, antiparasitic, anticancer/tumor and antibacterial activity. In this work, we tested five triazine Dyes for Lfcin B Affinity interactions using surface plasmon resonance (SPR) technology. Recombinant Lfcin B was expressed as a fusion protein with GST (Lfcin B-GST) by using the baculovirus expression vector system and the Dye-Sepharose matrices were assayed for Lfcin B-GST adsorption and subsequent elution. Red HE-3B and Yellow HE-4R Dyes were selected and immobilized on a Sepharose-4B matrix for further purification studies. The Yellow HE-4R-Sepharose matrix was specific for Lfcin B and allowed adsorption of Lfcin B-GST directly from the culture medium even at high salt concentration. This novel application of SPR to screen possible Dye–peptide interactions could be relevant to purify other peptides or proteins by using low-cost Dye-Affinity Chromatography.
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Improved hollow-fibre membranes for Dye-Affinity Chromatography.
Journal of separation science, 2005Co-Authors: Federico Javier Wolman, Osvaldo Cascone, Eduardo E. Smolko, Mariano GrasselliAbstract:Hollow-fibre membranes with different degrees of surface hydrophilicity were obtained by grafting mixtures of glycidyl methacrylate (GMA) and dimethyl acrylamide (DMAA) in various proportions, and Cibacron Blue F3G-A was attached to them through ammonia or glucamine spacers. Membrane hydrophilicity increased with the amount of dimethyl acrylamide in the grafted polymer. As the hydrophilicity increased the permeability decreased from 352 mL/cm2 min MPa for membranes grafted with GMA with ammonia spacer to 12.7 mL/cm2 min MPa for membranes grafted with GMA/DMAA 1/3 with glucamine spacer. Membranes grafted with GMA/DMAA 1/3 with ammonia spacer showed the best performance for BSA and lysozyme adsorption: maximum capacity was 15.3 +/- 2.2 mg BSA/mL membrane and 58.3 +/- 6.6 mg lysozyme/mL membrane while dissociation constants were 0.27 +/- 0.16 and 0.13 +/- 0.12 mg/mL, respectively. Over 80% of adsorbed proteins could be eluted with 2 M NaCl + 20% isopropanol in 20 mM sodium phosphate buffer, pH 7.0.
Osvaldo Cascone - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous purification and immobilization of soybean hull peroxidase with a Dye attached to chitosan mini-spheres
Biocatalysis and Biotransformation, 2017Co-Authors: Lautaro Fidel Bracco, Federico Javier Wolman, Gustavo Levin, Agustin A. Navarro Del Cañizo, María Victoria Miranda, Osvaldo CasconeAbstract:Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by Dye Affinity Chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditi...
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Lactoperoxidase purification from whey by using Dye Affinity Chromatography
Food and Bioproducts Processing, 2017Co-Authors: Nicolás Urtasun, María Fernanda Baieli, Daniela Belén Hirsch, María C. Martínez-ceron, Osvaldo Cascone, Federico Javier WolmanAbstract:Abstract Bovine lactoperoxidase is a glycoprotein present in milk, whey and colostrum, which might be used in dairy, cosmetic, pharmaceutical, veterinary and agricultural applications due to its broad antimicrobial activity. Here, we describe a novel process for bovine lactoperoxidase purification by using Dye Affinity Chromatography. Eighteen triazine Dyes were immobilized on Sepharose 6B and screened for their performance as possible ligands. Five of the Dye-Sepharose matrices showed over 90% adsorption of bovine lactoperoxidase directly from whey without any pretreatment using the batch mode, and were thus selected for further adsorption and elution studies. The highest elution degree was obtained using 20 mM acetate buffer, pH 5.0, 2 M NaCl, as the eluent for all the matrices. Whey processed using the Reactive Red 4-Sepharose matrix in batch mode showed the highest bovine lactoperoxidase purification yield (86.5 ± 3.8%), purification factor (46.1 ± 1.1), and a relative purity higher than 80% according to SDS-PAGE gel densitometry. Whey processed using packed-bed column mode showed lower yields and additional whey pretreatments were needed for dynamic processing. The interaction between bovine lactoperoxidase and Reactive Red 4-Sepharose matrix was characterized using Langmuir isotherm model. The K d value was 0.21 ± 0.03 mg/mL and the Q max was 32.21 ± 1.24 mg/g. The results presented here suggest the potential application of the Reactive Red 4-Sepharose matrix to one-step purification of bovine lactoperoxidase from whey.
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Isolation of lactoferrin from whey by Dye-Affinity Chromatography with Yellow HE-4R attached to chitosan mini-spheres
International Dairy Journal, 2014Co-Authors: María Fernanda Baieli, Nicolás Urtasun, Osvaldo Cascone, María Victoria Miranda, Federico Javier WolmanAbstract:Abstract Novel integrated chromatographic methods need to be developed for specific, low-cost protein purification from raw materials. Here, a process for bovine lactoferrin (Lf) isolation from sweet whey was developed using cross-linked chitosan mini-spheres with immobilised Yellow HE-4R Dye as a low-cost ligand. The maximum adsorption capacity was between 51.14 and 58.28 mg Lf g −1 matrix. In addition, the mini-spheres adsorbed around 95% of the Lf present in the sweet whey and eluted more than 80% of the adsorbed Lf. A yield of 77% with purity greater than 90% was achieved in only one purification step. The purification process was efficient for three consecutive cycles without regeneration steps.
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Triazinic Dye ligand selection by surface plasmon resonance for recombinant lactoferricin purification
Process Biochemistry, 2013Co-Authors: Nicolás Urtasun, María Fernanda Baieli, Osvaldo Cascone, Federico Javier Wolman, Pablo Nicolas Romasanta, Marisa M. Fernández, Emilio L. Malchiodi, María Victoria MirandaAbstract:Abstract Bovine lactoferricin (Lfcin B) belongs to the antimicrobial peptide family, which is the first line of defense against pathogens in many organisms. Lfcin B has important applications due to its antiviral, antifungal, antiparasitic, anticancer/tumor and antibacterial activity. In this work, we tested five triazine Dyes for Lfcin B Affinity interactions using surface plasmon resonance (SPR) technology. Recombinant Lfcin B was expressed as a fusion protein with GST (Lfcin B-GST) by using the baculovirus expression vector system and the Dye-Sepharose matrices were assayed for Lfcin B-GST adsorption and subsequent elution. Red HE-3B and Yellow HE-4R Dyes were selected and immobilized on a Sepharose-4B matrix for further purification studies. The Yellow HE-4R-Sepharose matrix was specific for Lfcin B and allowed adsorption of Lfcin B-GST directly from the culture medium even at high salt concentration. This novel application of SPR to screen possible Dye–peptide interactions could be relevant to purify other peptides or proteins by using low-cost Dye-Affinity Chromatography.
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Improved hollow-fibre membranes for Dye-Affinity Chromatography.
Journal of separation science, 2005Co-Authors: Federico Javier Wolman, Osvaldo Cascone, Eduardo E. Smolko, Mariano GrasselliAbstract:Hollow-fibre membranes with different degrees of surface hydrophilicity were obtained by grafting mixtures of glycidyl methacrylate (GMA) and dimethyl acrylamide (DMAA) in various proportions, and Cibacron Blue F3G-A was attached to them through ammonia or glucamine spacers. Membrane hydrophilicity increased with the amount of dimethyl acrylamide in the grafted polymer. As the hydrophilicity increased the permeability decreased from 352 mL/cm2 min MPa for membranes grafted with GMA with ammonia spacer to 12.7 mL/cm2 min MPa for membranes grafted with GMA/DMAA 1/3 with glucamine spacer. Membranes grafted with GMA/DMAA 1/3 with ammonia spacer showed the best performance for BSA and lysozyme adsorption: maximum capacity was 15.3 +/- 2.2 mg BSA/mL membrane and 58.3 +/- 6.6 mg lysozyme/mL membrane while dissociation constants were 0.27 +/- 0.16 and 0.13 +/- 0.12 mg/mL, respectively. Over 80% of adsorbed proteins could be eluted with 2 M NaCl + 20% isopropanol in 20 mM sodium phosphate buffer, pH 7.0.
