Immobilized Metal Affinity Membrane Research Articles

Preparation of a novel Zr(4+)-immobilized metal affinity membrane for selective adsorption of phosphoprotein.

In this study, a novel phosphate-Zr(4+) immobilized metal affinity membrane (IMAM) was prepared based on the surface initiated-atom transfer radical polymerization technique for the selective adsorption of phosphoprotein. The adsorption capacity and selectivity of the phosphate-Zr(4+) IMAM were evaluated by using the mixture of standard phosphoproteins (β-casein, ovalbumin) and nonphosphoproteins (bovine serum albumin and lysozyme) as model samples. The adsorption isotherms and competitive adsorption results demonstrated that the phosphate-Zr(4+) IMAM had higher binding capacity and selectivity for phosphoproteins over nonphosphoproteins. Moreover, the phosphate-Zr(4+) IMAM exhibited good re-usability and re-productivity. Finally, the phosphate-Zr(4+) IMAM was applied to separate phosphoprotein from real samples with high purity. Therefore, the as-prepared phosphate-Zr(4+) IMAM could be a promising affinity material for the efficient enrichment of phosphoprotein from complex bio-samples.

Antibody mediated rejection of renal transplantation – Clinicopathological correlation

Immobilized metal affinity membranes (IMAMs) were prepared by immobilizing copper ions on microporous regenerated cellulose membranes through different types of chelating agents (dentate and triazine dye). The resulting chelator utilization percentages were 95% for iminodiacetic acid, 56% for N,N,N-tris(carboxymethyl)ethylenediamine, 52% for Cibacron blue 3GA, and 140% for Cibacron red 3BA. On the other hand, triazine dyes were slightly superior to dentate chelators on metal ion utilization for protein adsorption. In batch single-protein adsorptions, the protein adsorption capacity decreased with increasing molecular size and number of accessible surface histidine residues [lysozyme>bovine serum albumin (BSA)>γ-globulin], while the binding strength order was the opposite (γ-globulin>BSA>lysozyme). Moreover, the proportions of specific and nonspecific bindings were evaluated by varying pH and salt concentration conditions. A large fraction of the adsorption capacity was found to come from the nonspecific interactions for the prepared IMAMs. Lastly, batch three-protein adsorptions were performed and weak adsorption competition was observed.

Adsorption of the Polyvinylidene Fluoride-Based Metal Affinity Membrane towards Bovine Serum Albumin

Three kinds of polyvinylidene fluoride (PVDF)-based immobilized metal affinity membranes (IMAM), namely, Cu (II)-IMAM, Co (II)-IMAM and Ni (II)-IMAM were prepared to recover bovine serum albumin (BSA) from the solutions. Adsorption of the aforementioned membranes towards BSA were studied with the presence of Ca (II) and PO43–. The adsorption performance of the membranes followed the order of Co (II)-IMAM > Cu (II)-IMAM > Ni (II)-IMAM. The existent PO43– exhibited a larger interference on BSA uptake than Ca (II).

Novel immobilized Cu+2 ion grafted cellophane membranes for affinity separation of His-Tag Chitinase

Two kinds of immobilized Cu+2 ion grafted cellophane membrane were prepared using methacrylic acid (MAA) and methyl methacrylate (MMA). The affinity grafted membranes were tested in separation of His-Tag Chitinase enzyme from BSA-protein mixture. PMAA grafted membranes show a higher affinity than PMMA grafted ones towards the separation of Chitinase enzyme. Both types of affinity membranes show almost the same desorption capability for both proteins with small advantage for PMAA grafted affinity membranes. No leakage of Cu+2 ions was detected during the protein elution process. The characters of the obtained Cu+2 immobilized grafted membranes recommended them as novel immobilized metal affinity membranes (IMAMs) for His-Tag protein separation and presenting a solution for the metal ion leakage problem; the main reason for limiting the use of metal ion affinity chromatography technique.

Facile Fabrication of Hydrophilic Nanofibrous Membranes with an Immobilized Metal–Chelate Affinity Complex for Selective Protein Separation

In this study, we report a facile approach to fabricate functionalized poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membranes as immobilized metal affinity membranes for selective protein separation. Hydrophilic PVA-co-PE nanofibrous membranes with controlled fiber sizes were prepared via a melt extrusion process. A chelating group, iminodiacetic acid (IDA), was covalently attached to cyanuric acid activated membrane surfaces to form coordinative complexes with metal ions. The prepared membranes were applied to recover a model protein, lysozyme, under various conditions, and a high lysozyme adsorption capacity of 199 mg/g membrane was found under the defined optimum conditions. Smaller fiber size with a higher immobilized metal ion density on membrane surfaces showed greater lysozyme adsorption capacity. The lysozyme adsorption capacity remained consistent during five repeated cycles of adsorption-elution operations, and up to 95% of adsorbed lysozyme was efficiently eluted by using a phosphate buffer containing 0.5 M NaCl and 0.5 M imidazole as an elution media. The successful separation of lysozyme with high purity from fresh chicken egg white was achieved by using the present affinity membrane. These remarkable features, such as high capacity and selectivity, easy regeneration, as well as reliable reusability, demonstrated the great potential of the metal-chelate affinity complex immobilized nanofibrous membranes for selective protein separation.

Functionalization and Characterization of Immobilized Metal Affinity Hydrogel Membranes

Previous studies have shown that functionalization of hydrogel membranes with metal affinity ligands increases the separation potential of the membranes for purification of biological systems. To achieve separation, histidines that are exposed on the proteins surface are attracted and attach to the metal ion within the IMA membrane matrix. The effectiveness of these membranes depends on several factors including, but not limited to, type of chelating group, the metal ion, and solution buffer. This paper is concerned with crosslinking of poly (vinyl) alcohol (PVA) with varied degrees of crosslinking that was subsequently functionalized with the immobilized metal affinity (IMA) group, that included a spacing element, (1, 4 Butanediol Diglycidyl ether (1,4 BDE)), chelator (NTA (Nitrilotriacetic acid)), and metal (Nickel (Ni2+). The impact of chelating ligand and glutaraldehyde concentration on the swelling and bound metal content was determined. Additionally, Fourier-transform infrared spectroscopy (FTIR) was used to examine the functional groups for the control (without metal attached) and affinity (with metal attached) membranes. Ultimately, this study suggests that crosslinking membranes with a higher glutaraldehyde percentage yielded a higher content of bound nickel.

Simultaneous purification and immobilization of d-hydantoinase on the immobilized metal affinity membrane via coordination bonds

This study constructs the immobilized metal affinity membrane (IMAM) via coupling of epichlorohydrin, iminodiacetic acid, and nickel ion on the regenerated cellulose membrane. The d-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein immobilized on the prepared IMAM. Various immobilization conditions were examined based on the yield of N-carbamoyl-d-p-hydroxyphenylglycine in batch reactions. The immobilization conditions were studied and the optimal conditions are as follows. By employing an IMAM with nickel ion of 155.5±5μmol/disc immersed in 0.1M Tris–HCl buffer pH 8 (with 0.8M sodium chloride) and immobilized time of 14h, a DHTase activity of 4.2±0.3U/disc was obtained. The immobilized DHTase membrane can achieve a larger pH and thermal tolerant range than that of free enzyme. Meanwhile, the stability test showed that 99% of enzyme activity could be retained after being repeated 15-times. The storage test also displayed 99% enzyme preservation after 7 weeks of storage.

Direct penicillin G acylase immobilization by using the self-prepared immobilized metal affinity membrane

The use of penicillin G acylase (PGA) requires efficient methods for immobilizing the enzyme to yield a biocatalyst with high activity and stability for industrial application. This study constructs the immobilized metal affinity membrane (IMAM) via a series of chemical reactions to sequentially couple epichlorohydrin (EPI), iminodiacetic acid (IDA), and copper ions on the regenerated cellulose membrane. The feasibility of direct PGA immobilization on IMAM was examined. Various conditions were tested for constructing IMAM for PGA immobilization. Under the optimal condition, a copper ion of 492.8 μmol/g with a PGA activity of 11.76 U/g was obtained for IMAM as compared to those for the commercial immobilized metal affinity resin (299.8 μmol/g and 2.99 U/g). The immobilized PGA membrane was stable in the pH range from 7 to 9 and for temperatures from 40 to 60 °C. In addition, 99% of the residual activity could be retained via a 16-times repeated use. Only little activity loss was observed after a 40 days of storage. This study also determines the kinetic parameters V max, K m, and the energy of activation ( E a) for the immobilized PGA membrane.

Exploring the complex effects of metal ions on d-hydantoinase purification with an immobilized metal affinity membrane

In this study, d-hydantoinase (DHTase) was purified using an immobilized metal ion affinity membrane (IMAM), on which the interactions between amino acid residues placed on the protein surface and the chelated metal ions located on the IMAM surface facilitate DHTase purification. Batch DHTase adsorption experiments showed that the adsorption capacity varied remarkably for IMAM with different metal ions. The maximum adsorption of DHTase (1.513 ± 0.12 mg) was reached when using Cu 2+ as the chelated ion, whereas the Co 2+ showed the highest activity with only small amounts of protein adsorption. The Mn 2+, Co 2+, Ni 2+, Fe 2+ and Fe 3+ additions showed a positive effect on DHTase activity. The addition of Cu 2+ showed a varied effect from the inhibition on original DHTase to the promotion on Ni-purified DHTase. The purification folds using IMAM chelated with Co 2+, Ni 2+, and Zn 2+ were in the range of six to seven. SDS–PAGE analysis showed that all of the IMAM-purified DHTase exhibited the same molecular weight, meaning DHTase adsorbed on IMAM was highly specific. The DHTase purified by different metal ions showed various levels of increased activity when adding the corresponding metal ions. The addition of Mn 2+ or Co 2+ displayed a dramatic increase (9- to 10-fold) in activity of DHTase purified by IMAM chelated with the same ion.

Papain Adsorption on Chitosan-Coated Nylon-Based Immobilized Metal Ion (Cu2+, Ni2+, Zn2+, Co2+) Affinity Membranes

A novel immobilized metal affinity membrane was prepared for papain adsorption in this article. Higher papain adsorption capacity between 43-67 mg/g was observed and the adsorption isotherm fitted the Freundlich equation. Experimental data were analyzed using two adsorption kinetic models. The pseudo-second-order kinetic model provided better correlation to the experimental results. A significant amount of the adsorbed papain was eluted by 1.0 M NaSCN at pH 5.0 for all affinity membranes. It was concluded that the novel chitosan-coated nylon-based immobilized metal ion affinity membrane could be applied for the large-scale isolation of papain without resulting in enzyme denaturation.

Preparation of the immobilized metal affinity membrane with high amount of metal ions and protein adsorption efficiencies

In this study, an approach to prepare immobilized metal affinity membrane (IMAM) with high metal ions and protein adsorption capacities was developed. In the process of coupling epichlorohydrin (EPI) to the regenerated cellulose membrane (RC membrane), NaOH concentration is found to be the most critical. With a lower NaOH concentration, only a minimal amount of EPI reacted to the RC membrane. When NaOH concentration was higher, the membrane was distorted, which caused a significant pressure drop in flow-through operation. To optimize the IMAM performance, an objective function was defined as the ratio of the model protein, penicillin G acylase (PGA), activity adsorbed on the membrane to the transmembrane pressure drop. According to the criterion, the optimal reaction conditions were found as follows: one RC membrane immersed in 20 ml, 1.4 M NaOH, 5 ml EPI and operated at 24 °C, 150 rpm for 14 h. Under this condition, the copper ions and PGA in IMAM were significantly increased to 75.5 ± 0.25 μmol/disc and 1.8 U/disc respectively. The adsorption for lysozyme on the prepared IMAM reached 1044 μg/cm 2, the highest in the literature.

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn 2+ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.

Development of an immobilized metal affinity membrane for penicillin g acylase purification

Development of an immobilized metal affinity membrane for penicillin g acylase purification

High-affinity sulfonated materials with transition metal counterions for enhanced protein separation in dual-layer hollow fiber membrane chromatography

High-affinity membrane materials have been successfully synthesized through a combination of the polymer sulfonation reaction with transition metal counterion exchange treatment. This type of promising materials were embodied for the first time with the aid of dual-layer hollow fiber technology for protein separation. Three types of immobilized metal affinity membranes (Cu 2+, Ni 2+ and Zn 2+ forms) were developed in this work and they all exhibited enhanced protein separation performance compared to the as-spun hollow fiber in H + form due to the strong affinity between transition metal counterions and target protein molecules. Ultimately, the high-purity target protein (>99%, w/w) could be achieved via the membrane in Cu 2+ form.

Immunoglobulin G adsorption behavior of l-histidine ligand attached and Lewis metal ions chelated affinity membranes

Immobilized metal affinity membranes were prepared by chelating Cu(II) and Fe(III) ions on poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA) membranes using l-histidine as a chelating ligand. To achieve this goal, the poly(HEMA-GMA) membrane was prepared via UV initiated photopolymerization. A spacer-arm (i.e., 1,6-diaminohexane) was introduced through the epoxy groups of the membrane (poly(HEMA-GMA)-SA). A chelating ligand (i.e., l-histidine amino acid) was covalently attached on the poly(HEMA-GMA) and/or poly(HEMA-GMA)-SA using glutaric dialdehyde as a coupling agent, poly(HEMA-GMA)-H and poly(HEMA-GMA)-SAH membranes, respectively. Then, Cu(II) and Fe(III) ions were chelated through poly(HEMA-GMA)-SAH membrane. The binding characteristics of human immunoglobulin G (IgG) to IMAC membranes and the selectivity of Cu(II) and Fe(III) ions to the IgG have been investigated from aqueous solution using l-histidine attached membrane (poly(HEMA-GMA)-SAH) as a control system. The experimental data was analyzed using two adsorption kinetic models, the pseudo-first-order and the pseudo-second-order, to determine the best-fit equation for the adsorption of IgG onto l-histidine incorporated and/or different metals ion immobilized affinity membranes. The first-order equation in the affinity membrane systems is the most appropriate equation to predict the adsorption capacity for all the tested adsorbents. Moreover, the effect of spacer-arm on the adsorption capacity was evaluated using poly(HEMA-GMA)-H membrane as a control system. The IgG binding order on the affinity membranes was poly(HEMA-GMA)-SAH-Cu(II) > poly(HEMA-GMA)-SAH-Fe(III) > poly(HEMA-GMA)-SAH > poly(HEMA-GMA)-H. Finally, the polarities and the surface free energies of the affinity membranes were determined by contact angle studies.

Effects of spacer arm on penicillin G acylase purification using immobilized metal affinity membranes

For studying penicillin G acylase (PGA) purification efficiency, various chain lengths of diamine ( n = 2–10) were applied as spacer arm to prepare the regenerated cellulose-based immobilized metal affinity membrane (IMAM). The effects of spacer arm length on water content of the IMAM, immobilized metal ion capacity, and batch PGA adsorption were investigated. The use of 1,10-diaminodecane led to the smallest water content of membrane and the maximum capacity of Cu 2+ (62.06 μmol/disc), whereas the optimal PGA adsorption capacity (2.6 IU/disc) was achieved with 1,8-diaminooctane. When applying the IMAMs with the spacer arm of 1,8-diaminooctane to the flow chromatographic experiments, a performance of 2.73-fold purification and 70.27% recovery was obtained. The employment of diamine spacer arm on the preparation of IMAMs resulted in higher PGA adsorption amount but less purification efficiency.

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 °C, 0.5 M NaCl, 32.04 μmol Cu 2+ per disk with 10 m M sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH 4Cl with 10 m M sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.

Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes

Immobilized metal affinity membranes (IMAMs) were prepared by immobilizing copper ions on microporous regenerated cellulose membranes through different types of chelating agents (dentate and triazine dye). The resulting chelator utilization percentages were 95% for iminodiacetic acid, 56% for N, N, N-tris(carboxymethyl)ethylenediamine, 52% for Cibacron blue 3GA, and 140% for Cibacron red 3BA. On the other hand, triazine dyes were slightly superior to dentate chelators on metal ion utilization for protein adsorption. In batch single-protein adsorptions, the protein adsorption capacity decreased with increasing molecular size and number of accessible surface histidine residues [lysozyme>bovine serum albumin (BSA)>γ-globulin], while the binding strength order was the opposite (γ-globulin>BSA>lysozyme). Moreover, the proportions of specific and nonspecific bindings were evaluated by varying pH and salt concentration conditions. A large fraction of the adsorption capacity was found to come from the nonspecific interactions for the prepared IMAMs. Lastly, batch three-protein adsorptions were performed and weak adsorption competition was observed.

Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.

Immobilized Metal Affinity Membrane Separation: Characteristics of Two Materials of Differing Preparation Chemistries

The focus of this research is the investigation of protein retention by two fabricated membrane modules which differ by a spacing element. Results indicate that both membranes have an equivalent equilibrium protein absorption capacity, but the breakthrough curves differ drastically. The data imply that traditional views regarding ligand accessibility during chromatographic separation merit special attention in a membrane format.