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Immobilized Metal Affinity Chromatography (immobilized + metal_affinity_chromatography)
Selected AbstractsImmobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Joseph Y. Fu Abstract Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20,mM HEPES, 240,mM NaCl, pH,7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations. Copyright © 2006 John Wiley & Sons, Ltd. [source] Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Shun Feng Abstract Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+ -IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of ,-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (,RPLC,nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12,,g mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with ,RPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample. [source] Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] On-line concentration of peptides and proteins with the hyphenation of polymer monolithic immobilized metal affinity chromatography and capillary electrophoresisELECTROPHORESIS, Issue 11 2005Lingyi Zhang Abstract An iminodiacetic acid (IDA)-type adsorbent is prepared at the one end of a capillary by covalently bonding IDA to the monolithic rods of macroporous poly(glycidyl methacrylate,co -ethylene dimethacrylate). Cu(II) is later introduced to the support via the interaction with IDA. By this means, polymer monolithic immobilized metal affinity chromatography (IMAC) materials are prepared. With such a column, IMAC for on-line concentration and capillary electrophoresis (CE) for the subsequent analysis are hyphenated for the analysis of peptides and proteins. The reproducibility of such a column has been proved good with relative standard deviations (RSDs) of dead time of less than 5% for injection-to-injection and 12% for column-to-column (n = 3). Through application on the analysis of standard peptides and real protein samples, such a technique has shown promising in proteome study. [source] A new human catalytic antibody Se-scFv-2D8 and its selenium-containing single domains with high GPX activityJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2010Junjie Xu Abstract Glutathione peroxidase (GPX) is a well-known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se-scFv-B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH-S-DNPBu was selected again by using the technology of phage display antibody library, and then scFv-2D8 was successfully expressed in soluble form and purified using Ni2+ -immobilized metal affinity chromatography. After being converted into selenium-containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se-scFv-B3. The heavy chain variable fragment of scFv-2D8 was also prepared and converted into selenium-containing protein using the same method. This selenium-containing single-domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single-domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity. Copyright © 2009 John Wiley & Sons, Ltd. [source] Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activityJOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008Rui Huo Abstract In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t -butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH,+,VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni2+ -immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0,3.0,mg of proteins from 1,L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288,U/µmol. Copyright © 2008 John Wiley & Sons, Ltd. [source] Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Mark R. Contarino Abstract As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1,mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15,nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain. Copyright © 2006 John Wiley & Sons, Ltd. [source] Solid-phase biotinylation of antibodies,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2004Elizabeth Strachan Abstract Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide,PEO2 biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS,PEO4 biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2,M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin,alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100,,g). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels. Copyright © 2004 John Wiley & Sons, Ltd. [source] New ,-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteinsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16-17 2008Genhu Lei Abstract A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, ,-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 ,mol 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+ -,-amino phenylalanine tetrazole,silica was found to be 1.21 ,mol/g at pH 6.0 and 1.20 ,mol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22×10,6 and 3.49×10,6 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. [source] Techniques for phosphopeptide enrichment prior to analysis by mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 1 2010Jamie D. Dunn Abstract Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29,54, 2010 [source] Serum biomarker profiling by solid-phase extraction with particle-embedded micro tips and matrix-assisted laser desorption/ionization mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2008Arti Navare One of the main challenges in high-throughput serum profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the development of proteome fractionation approaches that allow the acquisition of reproducible profiles with a maximum number of spectral features and minimum interferences from biological matrices. This study evaluates a new class of solid-phase extraction (SPE) pipette tips embedded with different chromatographic media for fractionation of model protein digests and serum samples. The materials embedded include strong anion exchange (SAX), weak cation exchange (WCX), C18, C8, C4, immobilized metal affinity chromatography (IMAC) and zirconium dioxide particles. Simple and rapid serum proteome profiling protocols based on these SPE micro tips are described and tested using a variety of MALDI matrices. We show that different types of particle-embedded SPE micro tips provide complementary information in terms of the spectral features detected for , -casein digests and control human serum samples. The effect of different sample pretreatments, such as serum dilution and ultrafiltration using molecular weight cut-off membranes, and the reproducibility observed for replicate experiments, are also evaluated. The results demonstrate the usefulness of these simple SPE tips combined with offline MALDI-TOF MS for obtaining information-rich serum profiles, resulting in a robust, versatile and reproducible open-source platform for serum biomarker discovery. Copyright © 2008 John Wiley & Sons, Ltd. [source] His-tagged protein purification by metal-chelate affinity extraction with nickel-chelate reverse micellesBIOTECHNOLOGY PROGRESS, Issue 4 2010Xiao-Yan Dong Abstract Di(2-ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X-45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R2. The Ni(II)-chelate reverse micelles were characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R2 was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni2+ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two-stage purification by the metal-chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Process intensification for the removal of poly-histidine fusion tags from recombinant proteins by an exopeptidaseBIOTECHNOLOGY PROGRESS, Issue 1 2010Wen-Hui K. Kuo Abstract This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their ApplicationCHINESE JOURNAL OF CHEMISTRY, Issue 7 2010Bolin Gong Abstract The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM-Asp) as chelating ligand and Ni2+ as center ion on the base of monodispersed, 3.0 µm non-porous monodisperse poly(glycidylmethacrylate- co -ethylenedimethacrylate) (PGMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min,1 by using the synthesized Ni2+ -CM-Asp-PGMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni2+ -CM-Asp-PGMA/EDMA column was further investigated for the rapid separation and purification of copper-zinc superoxide dismutase (Cu,Zn-SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory. [source] Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non-porous Beads and Their ApplicationCHINESE JOURNAL OF CHEMISTRY, Issue 5 2008Chun-Miao BO Abstract Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 µm monodisperse non-porous poly(glycidyl methacrylate- co -ethylenedimethacrylate) (PGMA/EDMA) beads. The retention behavior of proteins on the metal ion chelated columns loaded with copper(II), nickel(II) and zin(II) ion was studied. The effect of pH on the protein retention was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0. Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni2+ -IDA (iminodiacetic acid) packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked-IDA and Cu2+ -IDA columns, respectively. The purities of the purified trypsin and lysozyme were more than 92% and 95%, respectively. [source] | |||||||||||||