DNA adsorption via Co(II) immobilized cryogels

The separation and purification of important biomolecule deoxyribonucleic acid (DNA) molecules are extremely important. The adsorption technique among these methods is highly preferred as the adsorbent cryogels are pretty much used due to large pores and the associated flow channels. In this study, the adsorption of DNA via Co(II) immobilized poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [poly(HEMA-GMA)] cryogels was performed under varying conditions of pH, interaction time, initial DNA concentration, temperature, and ionic strength. For the characterization of cryogels; swelling test, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), surface area (BET), elemental and ICP-OES analysis were performed. L-lysine amino acid was chosen as Co(II)-chelating agent and the adsorption capacity of cryogels was determined as 33.81 mg DNA/g cryogel. Adsorption of pea DNA was studied under the optimum adsorption conditions and DNA adsorption capacity of cryogels was found as 10.14 mg DNA/g cryogel. The adsorption process was examined via Langmuir and Freundlich isotherm models and the Langmuir adsorption model was determined to be more appropriate for the DNA adsorption onto cryogels.     

Synthesis and characterization of supermacroporous cryogel with immobilized p-aminobenzenesulfonamide as affinity ligand for the purification of lactoperoxidase from whey

This study proposed the development of a monolithic supermacroporous affinity column for direct capture of lactoperoxidase, a glycoprotein present in milk, whey, and colostrum, with several applications due to its wide antimicrobial activity. A poly(acrylamide)-based cryogel was produced by radical co-polymerization of monomers in frozen aqueous solution and activated with p-aminobenzenesulfonamide as a ligand for specific interaction with the lactoperoxidase. The axial liquid dispersion coefficients at different liquid flow rates were determined by measuring residence time distributions using the tracer pulse-response method. The axial dispersion coefficient was low and the height equivalent to theoretical plate was not dependent on the flow velocity. The adsorptive capacity of affinity cryogel was studied as a function of flow velocity and the best condition was 0.9 cm/min. The response surface methodology was applied to optimize the capture of the enzyme, as a function of pH and salt concentration. Higher purification factor value was found at a salt concentration of 80 mmol/L and pH of 8.0 (p < 0.05). There was no influence of the variables under study on the yield (p > 0.05). The results indicated that affinity cryogel is a promising chromatography support for the use in high-throughput one-step purification of lactoperoxidase from whey.     

Hydrophobic cryogels for DNA adsorption: Effect of embedding of monosize microbeads into cryogel network on their adsorptive performances

As alternative hydrophobic adsorbent for DNA adsorption, supermacroporous cryogel disks were synthesized via free radical polymerization. In this study, we have prepared two kinds of cryogel disks: (i) poly(2‐hydroxyethyl methacrylate‐N‐methacryloyl‐l ‐tryptophan) [p(HEMA‐MATrp)] cryogel containing specific hydrophobic ligand MATrp; and (ii) monosize p(HEMA‐MATrp) particles synthesized via suspension polymerization embedded into p(HEMA) cryogel structure to obtain p(HEMA‐MATrp)/p(HEMA) composite cryogel disks. These cryogel disks containing hydrophobic functional group were characterized via swelling studies, Fourier transform infrared spectroscopy, elemental analysis, surface area measurements and scanning electron microscopy. DNA adsorption onto both p(HEMA‐MATrp) cryogel and p(HEMA‐MATrp)/p(HEMA) composite cryogels was investigated. Maximum adsorption of DNA on p(HEMA‐MATrp) cryogel was found to be 15 mg/g polymer. Otherwise, p(HEMA‐MATrp)/p(HEMA) composite cryogels significantly increased the DNA adsorption capacity to 38 mg/g polymer. Composite cryogels could be used repeatedly without significant loss on adsorption capacity after 10 repetitive adsorption–desorption cycles. Copyright © 2013 John Wiley & Sons, Ltd.     

Combined protein A imprinting and cryogelation for production of spherical affinity material

Cryogels have been demonstrated to be efficient when applied for protein isolation. Owing to their macroporous structure, cryogels can also be used for treating particle‐containing material, e.g. cell homogenates. Another challenging development in protein purification technology is the use of molecularly imprinted polymers (MIPs). These MIPs are robust and can be used repeatedly. The paper presents a new technology that combine the formation of cryogel beads concomitantly with making imprints of a protein. Protein A was chosen as the print molecule which was also be the target in the purification step. The present paper describes a new method to produce protein‐imprinted cryogel beads. The protein‐imprinted material was characterized and the separation properties were evaluated with regard to both the target protein and whole cells with target protein exposed on the cell surface. The maximum protein A adsorption was 18.1 mg/g of wet cryogel beads. The selectivity coefficient of protein A‐imprinted cryogel beads for protein A was 5.44 and 12.56 times greater than for the Fc fragment of IgG and protein G, respectively.     

Two-step polymerization approach for synthesis of macroporous surface ion-imprinted cryogels

Today, the surface imprinted polymers emerge in various fields as synthetic adsorbents gaining attention in a variety of application areas. In this study, Cu(II) ion surface imprinted poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA), cryogels were synthesized via modified two-step polymerization which is different from given in literature and the adsorption of Cu(II) ion from aqueous solution was investigated batch wise. In this respect, the method applied in this study is new in the literature despite heavy metal removal studies reported. The polyethyleneimine (PEI) molecule was used in polymeric structure as a ligand. The poly(HEMA-GMA) cryogels prepared was characterized via Fourier transform infrared spectroscopy (FTIR), inductively coupled plasma optical emission spectrometry (ICP-OES), elemental analysis, scanning electron microscopy (SEM) and the micro-computed tomography (μCT).     

Magnetic dye affinity beads for the adsorption of beta-casein

Casein is well known as a good protein emulsifier and beta-casein is the major component of casein and commercial sodium caseinate. Dye affinity adsorption is increasingly used for protein separation. beta-Casein adsorption onto Reactive Red 120 attached magnetic poly(2-hydroxyethyl methacrylate) (m-PHEMA) beads was investigated in this work. m-PHEMA beads (80-120 microm in diameter) were produced by dispersion polymerization. The dichlorotriazine dye Reactive Red 120 was attached covalently as a ligand. The dye attached beads, having a swelling ratio of 55% (w/w) and carrying different amounts of Reactive Red 120 (9.2 micromol . g(-1)-39.8 micromol . g(-1)), were used in beta-casein adsorption studies. The effects of the initial concentration, pH, ionic strength and temperature on the adsorption efficiency of dye attached beads were studied in a batch reactor. The non-specific adsorption on the m-PHEMA beads was 1.4 mg . g(-1). Reactive Red 120 attachment significantly increased the beta-casein adsorption up to 37.3 mg . g(-1). More than 95.4% of the adsorbed beta-casein was desorbed in 1 h in a desorption medium containing 1.0 M KSCN at pH 8.0. We concluded that Reactive Red 120 attached m-PHEMA beads can be applied for beta-casein adsorption without significant losses in the adsorption capacities.     

Development and screening of epoxy‐spacer‐phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide‐expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost‐effective one‐step chromatographic set‐up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di‐amino‐alkanes (C₂–C₁₀), and branched polyethyleneimine spacers with different molecular weights (800 Da–10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15‐mer and a cyclic 6‐mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.     

Composite cryogel with immobilized concanavalin A for affinity chromatography of glycoproteins

Composite cryogels containing porous adsorbent particles were prepared under cryogelation conditions. The composites with immobilized concanavalin A (Con A) were used for capturing glycoproteins. Adsorbent particles were introduced into the structure in order to improve the capacity and to facilitate the handling of the particles. The monolithic composite cryogels were produced from suspensions of polyvinyl alcohol particles and porous adsorbent particles and cross‐linked under acidic conditions at sub‐zero temperature. The cryogels were epoxy activated and Con A was immobilized as an affinity ligand. Binding and elution of horseradish peroxidase (HRP) was studied in batch experiment and in a chromatographic setup. Increasing adsorbent concentration in composite cryogels will increase ligand density, which therefore enhances the amount of bound HRP from 0.98 till 2.9 (milligram enzyme per milliliter of gel) in the chromatographic system. The material was evaluated in 10 cycles for binding and elution of HRP.     

Highly Sensitive Determination of Concanavalin A Lectin Based on Silver-Enhanced Electrogenerated Chemiluminescence of Luminol

A highly sensitive bioassay based on silver-enhanced luminol electrogenerated chemiluminescence (ECL) is reported for the determination of concanavalin A lectin. A gold electrode modified with the mixed self-assembled monolayer of thiolated mannoside and mercaptohexanol was used to selectively capture a target lectin, concanavalin A, through the specific interaction between mannoside and concanavalin A. Mannoside-functionalized gold nanoparticles were further introduced to the opposite binding sites of the tetrameric concanavalin A to form a sandwich-type complex. Silver enhancement step was performed to coat the surface of mannose-stabilized gold nanoparticles with silver. The deposited silver was dissolved in an acidic solution and further neutralized. The resulting silver ions were finally detected with luminol electrogenerated chemiluminescence, in which the silver ions greatly enhanced the chemiluminescence intensity. The present electrogenerated chemiluminescence bioassay detected concanavalin A from 0.190 to 10.0?µg/mL (r²?=?0.999) with a detection limit of 0.146?µg/mL (signal to noise ratio?=?3), which is much lower compared to previously reported methods such as microgravimetry, surface plasmon resonance, and colorimetry. Furthermore, the present bioassay showed good selectivity over possible interfering lectin proteins.     

Application of Lectin Microarrays for Biomarker Discovery

Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein-, cell-, and tissue-specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high-throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers.     

N-glycosylation proteome of endoplasmic reticulum in mouse liver by ConA affinity chromatography coupled with LTQ-FT mass spectrometry

Glycosylation is the most versatile and one of the most significant protein post-translational modifications. It is generally classified into three categories according to the amino acid to which the glycan is attached: N-glycosylation, O-glycosylation and C-glycosylation. Synthesis of N-glycoproteins occurs in the rough endoplasmic reticulum (rER), and all N-glycoproteins synthesized in rER have uniform glycan endings with mannose (Man) and glucose (Glc). A systematic strategy was developed to comprehensiv...     

High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G

High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.     

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

Solution‐blown nylon 6–chitosan core–shell nanofiber for highly efficient affinity adsorption

A facile spinning‐based strategy was developed to fabricate chitosan (CS) surface nanofiber‐based affinity membranes for protein adsorption. The core–shell nanofiber mat of nylon 6–CS was prepared via coaxial solution blowing process. The nanofibers have a diameter range of 60–300 nm. The core–shell structure was confirmed by transmission electron microscopy, and CS was observed as a thin layer that uniformly adhered to the core. The dye ligand of cibacron blue F3GA (CB F3GA) was further covalently immobilized on the nanofibers with a content of 425 µmol/g. The pristine and CB F3GA‐attached mats were studied in protein adsorption. High bovine serum albumin adsorption capacities of 91.9 and 219.6 mg/g were obtained for pristine and CB F3GA‐attached mats, respectively. Given its properties of high flux rate and low pressure drop, CB F3GA‐attached nylon 6–CS nanofiber mat meets the requirements of highly effective affinity membrane chromatography. Copyright © 2016 John Wiley & Sons, Ltd.     

Dyes with high affinity for polylactide

Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes. The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

Boronate affinity adsorption of cis‐diol‐containing biomolecules in nonaqueous solvent

Boronate affinity has attracted much attention in recent years. It has been broadly used for selective isolation and enrichment of cis‐diol‐containing molecules. Conventionally, the cis‐diols are adsorbed in mild alkaline aqueous solutions. In this work, for the first time, we found that boronate affinity adsorption could also be performed in nonaqueous solvent at nonbasic pH. Cis‐diol‐containing compounds present in herbal medicines were used for the adsorption test. The results indicated that all compounds obtained higher recoveries in the organic solvents (methanol, acetonitrile, ethyl acetate) compared with alkaline buffer. The adsorption of vicinal cis‐diol‐containing molecules in organic solvents could be accomplished rapidly, with high selectivity and high recoveries (>80%). These results shed light on the possibility of boronate affinity adsorption in nonaqueous solvents. The results are very important for the isolation and enrichment of cis‐diols, which have poor solubility in water, especially for those in herbal medicines. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography

We developed a simple high-performance liquid chromatography assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2-aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha-mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase-treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal-mannose binding to the column and comparable data trending with high mannose levels by 2-aminobenzamide assay confirmed that terminal-mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high-mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high-mannose content in samples compared with traditional glycan profiling by 2-aminobenzamide or mass spectrometry-based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two-dimensional analysis, high-mannose data could be relayed to the manufacturing team in less than 30 min, allowing near-real-time monitoring of high-mannose levels in the cell culture process.