Dye-ligand Affinity Chromatography Research Articles

Cibacron Blue F3GA ligand dye-based magnetic silica particles for the albumin purification.

Dye-ligand affinity chromatography is among the increasingly popular affinity chromatography based on molecular recognition for the purification of albumin. This study focuses on the binding of Cibacron Blue F3GA ligand dye with magnetic silica particles and purification by separation. Mono-disperse silica particles with bimodal pore size distribution were employed as a high-performance adsorbent for human serum albumin (HSA) protein purification under equilibrium conditions. The synthesized ligand-dye affinity based magnetic silica particles were characterized by electron spin resonance, Fourier-transform infrared spectroscopy, scanning electron microscopy, vibrating sample magnetometer, elemental analysis, and dispersive X-ray analysis. The HSA purification performance of the proposed material in the presence of a magnetic field was relatively investigated using magnetic-based particles with similar morphologies. The maximum adsorption capacity for HSA in an artificial plasma medium was defined as 48.6 mg/g magnetic silica particle. By using the designed magnetic silica particles, 1.0 M NaCl solution was successfully utilized for obtaining quantitative desorption with HSA. However, continued HSA purification performances of magnetic-based particles were significantly lower concerning the ligand-dye magnetic silica particles. The purity of the removed albumin was about 97%. The magnetic silica particles could be utilized many times without decreasing their protein adsorption capacities remarkably.

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

Human serum albumin depletion based on dye ligand affinity chromatography via magnetic microcryogels

One of the primary purposes of proteomic studies is to analyze the proteins in the blood to be considered as biomarkers. Albumin, which constitutes the majority of total serum proteins, complicates the discovery of low-density proteins that are important for the diagnosis of diseases. Based on this, an alternative approach for albumin depletion was developed in this study by covalently attached Cibacron Blue 3GA (CB) to magnetic microcryogels. After detailed characterization of CB attached magnetic microcryogels synthesized via a microstencil array chip, albumin adsorption studies were performed to examine the optimum depletion conditions. In the presented study, the maximum albumin adsorption capacity (Qmax) was calculated as 149.25 mg/mL in pH 5.0 acetate buffer solution, which is the optimum pH value for albumin. Experimental studies have demonstrated that CB-attached magnetic microcryogels can be reused without loss of performance for albumin depletion after 10 adsorption-desorption cycles.

Synthesis and Evaluation of Dye-Ligand Affinity Adsorbents for Protein Purification.

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity toward a large number of proteins. They are commercially available, inexpensive, stable and can easily be immobilized. Significant factors that contribute to the successful operation of a dye-ligand chromatography include matrix type, dye-ligand density, adsorption along with elution conditions and flow rate. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a givendye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris on Cibacron Blue 3GA-Sepharose affinity adsorbent is given as an example.

Method for efficient soluble expression and purification of recombinant human interleukin-15

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.

Direct recovery of malate dehydrogenase from highly turbid yeast cell homogenate using dye-ligand affinity chromatography in stirred fluidized bed

Dye-ligand affinity chromatography in a stirred fluidized bed has been developed for the rapid recovery of malate dehydrogenase (MDH) from highly turbid baker's yeast cell homogenate in a single step. The most suitable dye, namely Reactive Orange 4, in its optimal immobilized concentration of 8.78 mg/mL was immobilized onto high-density STREAMLINE matrix. To further examine optimal adsorption and elution conditions, the enzyme recovery operation was carried out using unclarified cell homogenates in stirred fluidized bed system. Aiming to develop a non-specific eluent, namely NaCl, to effectively elute the MDH adsorbed, direct recovery of MDH from highly turbid cell homogenate (50% w/v) in a stirred fluidized bed adsorption system was performed. The proposed system successfully achieved a recovery yield of 73.6% and a purification factor of 73.5 in a single step by using 0.6 M NaCl as an eluent at a high liquid velocity of 200 cm/h.

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.

Affinity monolith chromatography: A review of general principles and applications.

Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological-binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose, and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined.

Застосування барвник-лігандної хроматографії для очищення фактора VIII зсідання крові

The problem of processing of donor plasma for creating an effective medications is important. Nowadays a big number of methods for fractionation of plasma separation and purification components, especially coagulation factors and fibrinolysis were described. The methods of biospecifical chromatography are especially attractive. The chromatographic purification of factor VIII is a trouble due to potential risks of activation and instability in the presence of plasma proteases. The anion exchange, gel-filtration and affinity chromatography are widely used for purification of coagulation factor VIII. The synthetic active triazine dyes have a number of advantages as ligands. We explored the possibility of using dye-ligand affinity chromatography on silica carriers for the isolation and purification of clotting factor VIII. The results of studies of sorption this factor with different chromatographic sorbents and the method of its purification from cryoprecipitate by binding of additional proteins on Diasorb aminopropyl with immobilized triazine active dyes are described. As a result of this work, we have selected a group of sorbents are the best for purification of factor VIII of blood coagulation: Diasorb-Active scarlet damask 4GT, Diasorb-Procion gelb M4R, Diasorb-Procion blue HB, Diasorb-Procion blue MXR and Diasorb-Active bright blue K. The purification of factor VIII occured due to the phenomenon of negative affinity chromatography. The specific activity of factor increased approximately tenfold.

Interaction Mechanism in Dye‐Ligand Affinity Chromatography

Chemie Ingenieur TechnikVolume 86, Issue 9 p. 1617-1618 VortragFree Access Interaction Mechanism in Dye-Ligand Affinity Chromatography J. Liang, Corresponding Author J. Liang juan.liang@tu-harburg.de Hamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, GermanyHamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, Germany===Search for more papers by this authorProf. Dr.-Ing. G. Fieg, Prof. Dr.-Ing. G. Fieg Hamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, GermanySearch for more papers by this author J. Liang, Corresponding Author J. Liang juan.liang@tu-harburg.de Hamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, GermanyHamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, Germany===Search for more papers by this authorProf. Dr.-Ing. G. Fieg, Prof. Dr.-Ing. G. Fieg Hamburg University of Technology, Institute of Process and Plant Engineering, Schwarzenbergstraße 95, D-21073 Hamburg, GermanySearch for more papers by this author First published: 28 August 2014 https://doi.org/10.1002/cite.201450026AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume86, Issue9Special Issue: ProcessNet-Jahrestagung 2014 und 31. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2014Pages 1617-1618 RelatedInformation

Ein Vergleich von Modellen für Interphasenkräfte in Euler‐Euler CFD‐Simulationen von Blasensäulen

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Ing. Tech. 2014, 86, No. 9, 1615–1632 Dye-ligand affinity chromatography is widely utilized for protein separation and purification. Immobilized textile trazine dyes, particularly Cibacron blue, have been applied as affinity chromatography tools for a long time. However, its interaction with a large number of seemingly unrelated proteins inevitably compromises its protein-binding specificity. This endows Cibacron blue with a serious drawback. To cope with the lack of specificity of textile dyes, a better understanding of the binding and specific mechanisms between dye-ligand and different proteins is required. For a detailed investigation into the interaction between Cibacron blue and proteins, molecular dynamics simulations were applied to study the interactions of Cibacron blue with two different proteins, serum albumin and hemoglobin. Allatom models of these affinity interaction systems were employed for a precise study. The interaction as well as the binding sites of proteins are analyzed and discussed in detail. For a better understanding of the specific mechanism, the simulations of these two proteins are compared. The simulation results are combined with the former experimental results to obtain a comprehensive understanding, which is important for the design of new dye-ligands as well as for optimization of dye-ligand affinity chromatography.

Affinity monolith chromatography: a review of principles and recent analytical applications

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.

Dye Ligand Epoxide Chitosan/Alginate: A Potential New Stationary Phase for Human IgG Purification

Cibacron Blue F3GA immobilized as ligand onto epoxide chitosan/alginate composite was prepared and evaluated for the purification of human immunoglobulin G (IgG) isolated from human serum by dye-ligand affinity chromatography. The effect of buffer pH on IgG adsorption, kinetic and isothern adsorption of high-purity IgG were studied by batch adsorption. The adsorption isotherm data were well fitted (using 25 mmol/L sodium phosphate buffer at pH 6.0) by the Langmuir-Freundlich model with a maximum adsorption capacity of 99 mg/g, apparent dissociation constant of 1.2 x 10-6 mol/L and n equal to 4.86. Fixed-bed experiments were performed using high-purity IgG, achieving a recovery rate of 53%. Human serum diluted ten times was also injected into the column. An enrichment of IgG in the elution fraction with traces of human serum albumin was observed. According to the study results, this material can be applied in the purification of human IgG under optimized conditions.

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands.Electronic supplementary materialThe online version of this article (doi:10.1007/s10969-012-9124-8) contains supplementary material, which is available to authorized users.

One step purification of biological active human interleukin-2 protein produced in yeast (Pichia Pastoris)

Pharmacological importance of recombinant human interleukin-2 protein has increased the demand to establish effective, reliable and cost effective chromatography method for its production and purification on large scale. One step mimetic ligand affinity chromatography method for purification of mutated human recombinant interleukin-2 (mrhIL-2), from Pichia Pastoris is described with higher yield then reported before. The mrhIL-2 was expressed extracellularly under methanol inducible AOX1 promoter of P. pastoris. Extracellular expression of mrhIL-2 in the culture supernatant was ~210 mg/L. Cell free culture supernatant containing mrhIL-2 protein was concentrated and buffer exchanged by diafiltration by tangential flow filtration system. Different mimetic legends column from sigma were screened for efficient binding of mrhIL-2 in culture supernatant. Maximum binding was observed with Mimetic Blue SA P6XL and least with Mimetic Green 1 A6XL. One step Dye ligand affinity chromatography method was developed by using Mimetic Blue SA P6XL ligand for high level purification of mutant of interleukin-2. Final yield of purified protein was 115 mg/L and purity of 97%. The interleukin-2 protein prepared by this protocol was found to be monomeric based on SDS-PAGE, western blot and HPLC analysis. Purified protein was biological active as checked by cell proliferation assay. Key words: Interleukin-2, Pichia pastoris, Dye ligand affinity chromatography.

Development of the magnetic beads for dye ligand affinity chromatography and application to magnetically stabilized fluidized bed system

Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe 3O 4 nano-powder. Cibacron Blue F3GA was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/Cibacron Blue F3GA beads (100–140 μm in diameter) carrying 68.3 μmol Cibacron Blue F3GA per gram polymer were used for β-casein adsorption studies. Adsorption studies were performed under different conditions in a batch system (i.e., pH, β-casein initial concentration, temperature, and ionic strength) and then in a magnetically stabilized fluidized bed (MSFB) system. The swelling ratio of the mPHEMA was 62.1%. The maximum adsorption capacity for batch system was 20.2% lower as compared to the value obtained in MSFB. The mPHEMA/Cibacron Blue F3GA beads could be repeatedly applied for β-casein adsorption without significant losses in the adsorption capacity.

Discriminatory protein binding by a library of 96 new affinity resins: A novel dye-affinity chromatography tool-kit

Initial acceptance of Cibacron Blue 3G-A ® based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A ® and also by varying spacer lengths for immobilization. The library was tested in a batch binding and elution mode using seven different proteins – four Aspergillus enzymes namely, NADP-glutamate dehydrogenase, laccase, glutamine synthetase and arginase, bovine pancreatic trypsin and the two serum proteins human serum albumin and immunoglobulin G. Unique binding patterns were observed for each of them indicating that the library displayed discriminatory interactions. The significance of spacer length in the interaction with proteins was discernable. Trypsin interacted best with affinity resins that had no spacer. It was possible to resolve IgG and HSA from a mixture using a combination of resins. There was a good spread of HSA binding capacity in the 96 affinity resins. While some showed better HSA binding capacity than the commercial CB3GA-based matrix, a few with lower capacity were also observed. Subsequent to an initial screen, one affinity resin (CR-017) could be used to enrich Aspergillus terreus NADP-GDH from crude cell extracts. The efficacy of this dye-affinity resin was rationalized by characterizing NADP-GDH inhibition kinetics with the corresponding free dye ligand. In the sum, the library provides a set of dye-ligand affinity matrices with a potential for use in high throughput screening for protein purification.

Novel column-based protein refolding strategy using dye-ligand affinity chromatography based on macroporous biomaterial

A novel column-based chromatographic protein refolding strategy was developed using dye-ligand affinity chromatography (DLAC) based on macroporous biomaterial. Chitosan–silica (CS–silica) biomaterial with macroporous surface was used as the supporting matrix for the preparation of the DLAC material. The dye-ligand Cibacron Blue F3GA (CBF) was selected as affinity handle and could be covalently immobilized to form dye-ligand affinity adsorbent (CBF–CS–silica) using the reactivity of NH 2 on CS–silica biomaterial. After the model protein catalase was denatured with 6 mol/L urea, the denaturant could be rapidly removed and catalase could be successfully refolded as facilitated by the adsorption of CBF–CS–silica. The urea denaturation process and the elute condition for the chromatographic refolding were optimized by measuring tryptophan fluorescence and activity of catalase. The refolding performance of the proposed DLAC was compared with dilution refolding. The protein concentration during the proposed chromatographic refolding increased by a factor of 20 without reducing the yield achieved as compared to dilution refolding. The column-based protein refolding strategy based on dye-ligand affinity chromatography with porous biomaterial being matrix possessed potential in chromatographic refolding of protein.

Affinity monolith chromatography

The combined use of monolithic supports with selective affinity ligands as stationary phases has recently given rise to a new method known as affinity monolith chromatography (AMC). This review will discuss the basic principles behind AMC and examine the types of supports and ligands that have been employed in this method. Approaches for placing affinity ligands in monoliths will be considered, including methods based on covalent immobilization, biospecific adsorption, entrapment, and the formation of coordination complexes. Several reported applications will then be presented, such as the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, and biomimetic chromatography. Other applications that will be discussed are chiral separations and studies of biological interactions based on AMC.

Biochemical Characterization and Crystal Structure of Synechocystis Arogenate Dehydrogenase Provide Insights into Catalytic Reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.