New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.     

Immobilized metal ion affinity chromatography of proteins on IDA-Fe3+

Several proteins, selected for their varied isoelectric points within pH range ca. 4 to ca. ll, bind to immobilized (chelated to iminodiacetate) ferric ion, IDA-Fe³⁺, when applied in a 50 mM buffer, pH 6.0. These proteins can be displaced from IDA-Fe³⁺ columns by an increase of pH from 6 to 8, an increase of sodium chloride from 0 to 1 Malar and, in particular, by both. Apotransferrins, in contrast to other proteins, are able to scavenge Fe³⁺ ion from IDA-Fe³⁺. Interestingly, two proteins, both quite acidic, behave quite differently on IDA-Fe³⁺: α₁-acid glycoprotein (sialic acid) does not bind, whereas phosvitin (phosphate 1) binds avidly. IDA-Fe³⁺ sorbent, due to its unusual sorptive properties, represents a new addition of particular significance to the family of chromatographic sorbents available for protein purification.     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。     

Study of pepsin phosphorylation using immobilized metal affinity chromatography

The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.     

Separation of chitosan oligomers by immobilized metal affinity chromatography

A novel approach for chitosan oligosaccharide (COS) separation by immobilized metal affinity chromatography (IMAC) based on the differences in the interactions of chelated copper (II) ions with various COS (dimers, trimers, tetramers) is described. Polyhydroxylic chromatographic supports (agarose CL-6B and silica) were functionalized with various chelating functions such as iminodiacetate (IDA), carboxymethyl-aspartate (CM-Asp) and tris(carboxymethyl)ethylenediamine (TED). The COS retention capacities of the columns were between 2 and 6 mg/cm(3), depending on the chelating group. The COS were separated and/or enriched up to 95% for dimer and trimer and 90% for the tetramer, with yields of 60-95%.     

Separation and evaluation of soybean protein hydrolysates prepared by immobilized metal ion affinity chromatography with different metal ions

Metal ion affinity chromatography is widely used to purify peptides on the basis of the dissimilarities of their amino acids. However, researchers are interested in the separation differences between different metal ions in this method. In our study, four kinds of commonly used metal ions are compared by the amount of immobilized metal ion on iminodiacetic acid-Sepharose and binding amount of soybean peptide to immobilized iminodiacetic acid-Mn(+) adsorbents and evaluated by high-performance liquid chromatography (HPLC) profiles. The results show that due to the different adsorption behaviors of metal ions, the binding ability order of soybean protein peptide on the column should be Fe(3+) > Cu(2+) > Zn(2+) > Ca(2+). The HPLC profiles show that peptides adsorbed by four kinds of metal ions display similar strong hydrophobic characteristics.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).     

固定化金属离子亲和发光二氧化硅纳米粒子的制备及其用于磷酸化蛋白免疫印迹标记

发展了一种能够识别磷酸化蛋白的固定化金属离子亲和发光二氧化硅纳米粒子用于免疫印迹(Western Blot)磷酸化蛋白的标记.首先通过反相微乳液St?ber方法合成了掺杂异硫氰酸荧光素硅烷化衍生物的发光二氧化硅(FITC@SiO2)球形纳米粒子,粒子平均粒径为60 nm.然后通过共聚反应在FITC@SiO2纳米粒子表面...     

Fluorescence study of an immobilized ligand—metal ion complex

A ligand known to form a fluorescent complex with aluminum ion was immobilized on silica gel. The immobilization sequence was verified by cross-polarization magicangle spinning n.m.r. spectroscopy and diffuse reflectance u.v. spectroscopy. The solid-state fluorescence of the immobilized ligand complexed with aluminum ion was similar to the fluorescence of a solvated complex of a model ligand. The potential to eliminate possible interfering species by isolating the complex from solution was demonstrated.     

Purification of S-oxynitrilase from Sorghum bicolor by immobilized metal ion affinity chromatography on different carrier materials.

The purification of the hydroxynitrile lyase (EC 4.1.2.11, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.     

Poly(hydroxyethyl methacrylate) membranes: as a hydrogel support for use in immobilized metal affinity chromatography

Lysozyme adsorption onto a dye ligand (Procion Red HE-3B) immobilized and Cu(II) incorporated poly(2-hydroxyethylmethacrylate) (pHEMA) membrane, were investigated. The membranes were prepared by UV initiated photopolymerization of HEMA in the presence of an initiator (α-α′-azoisobutyronitrile; AIBN). The amount of immobilized dye on the membrane was 112.2 μmol g⁻¹. Lysozyme adsorption on to these membranes from aqueous solutions containing different amounts of lysozyme at different pH was investigated in batch system. Lysozyme adsorption capacity of the dye-ligand immobilized membrane was 45.6 mg g⁻¹. Lysozyme adsorption capacity of the Cu(II) incorporated membranes (112.3 mg g⁻¹) was greater than that of the Procion Red HE-3B immobilized membranes. The non-specific adsorption of lysozyme on the pHEMA membranes was 0.14 mg g⁻¹. More than 97% of the adsorbed lysozyme were desorbed in 60 in the desorption medium containing 1.0 M KSCN at pH 8.0.     

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.     

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.     

Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) was investigated as a method of recovery for green fluorescent protein (GFPuv). It was found that in the absence of genetic modification to enhance metal affinity, GFPuv displayed strong metal affinity to Cu(II) and Ni(II), and weak or negligible affinity to Zn(II) and Co(II). Changes in the mobile phase NaCl concentration during Ni(II)-IMAC strongly affected purity and yield of GFPuv, with fine resolution under higher NaCl concentrations. Finally, IMAC via Cu(II) and Zn(II) with intervening diafiltration was used to recover GFPuv with high yield and purity.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.