Nucleic acid separations utilizing immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is widely used for protein purification, e.g., in the isolation of proteins bearing the well-known hexahistidine affinity tag. We report that IMAC matrixes can also adsorb single-stranded nucleic acids through metal ion interactions with aromatic base nitrogens and propose that metal affinity technologies may find widespread application in nucleic acid technology. Oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. The affinity of yeast RNA for IDA-chelated metal ions decreases in the following order: Cu(II), Ni(II), Zn(II), and Co(II). Adsorption isotherms for 20-mer oligonucleotide homopolymers show that purines are strongly favored over pyrimidines and that double-stranded duplexes are not bound. IMAC columns have been used to purify plasmid DNA from E. coli alkaline lysates, to purify a ribozyme, to remove primers and imperfect products from PCR reactions, and to separate 20-mer oligonucleotide duplexes containing centered single-base mismatches. Potential further applications include SNP scoring, hybridization assays, and the isolation of polyadenylated messenger RNA.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

Modeling of non-ideal displacement separation in immobilized metal ion affinity chromatography

To predict complex behavior in protein displacement systems of immobilized metal ion affinity chromatography (IMAC), numerical simulation of non-linear chromatography was developed and compared with the ideal solution of the model. The theoretical and experimental results demonstrate that the IMAC model can be successfully employed in predicting induced mobile phase modifier gradients and complex behavior in metal affinity displacement chromatography. The solute movement analysis is able to predict the displacement separation well characterized by the intersections of the operation line under the induced mobile phase modifier and effective displacer concentrations.     

Screening of suitable immobilized metal chelates for adsorption of monoclonal antibodies against mutant amidase from Pseudomonas aeruginosa

The chromatographic behaviour of monoclonal antibodies (MAbs) of IgM class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated. The effect of ligand concentration, the length of spacer arm and the nature of metal ion were investigated on immobilized metal ion affinity chromatography (IMAC). MAbs against mutant amidase adsorbed to Cu (II), Ni (II), Zn (II), Co (II) and Ca (II)-IDA agarose columns. The adsorption of MAbs onto immobilized metal chelates was pH dependent because an increase in the binding of MAbs was observed as the pH was raised from 6.0 to 8.0. The adsorption of MAbs to metal chelates was due to coordination of histidine residues which are available in the 3rd constant domain of heavy chain (CH3) of immunoglobulins since the presence of imidazole in the equilibration buffer abolished the adsorption of MAbs to the column packed with commercial IDA-Zn(II) agarose at pH 8.0. The combination of tailor-made stationary phases for IMAC and a correct choice of the adsorption conditions permitted to design a one-step purification procedure for MAbs of IgM class. Culture supernatants containing MAbs of IgM class against mutant amidase (T103I) were chromatographed by IMAC Co (II) column at pH 8.0. The results strongly suggest that one-step purification of MAbs of IgM class by IMAC is a cost-effective and process-compatible alternative to the other purification procedures.     

固定化金属螯合亲和膜纯化重组抗菌肽研究

用自行制备的固定化金属螯合亲和膜对N端含六个组氨酸标记的重组抗菌肽进行了分离纯化,并较好地解决了金属离子泄露问题.实验表明,固定化金属螯合亲和膜性能优于传统琼脂糖凝胶介质,完全适用于分离纯化含有多组氨酸标记的重组蛋白质.     

Immobilized metal ion affinity chromatography

This article describes the technique of immobilized metal ion affinity chromatography (1MAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chclation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.     

Selective separation of caseinophosphopeptides through immobilized metal ion affinity chromatography: effects of pH values, salt concentrations and degree of phosphorylation

A method based on immobilized metal ion affinity chromatography (IMAC) using a CS-IDA-Cu(II) gel is described for the separation of caseinophosphopeptides (CPPs) from a casein hydrolysate. According to the amino acid sequence and the degree of phosphorylation, operating parameters, such as pH values, salt concentrations for selective purification of different CPPs can be designed.     

Immobilized metal ion affinity chromatography: a review on its applications

After 35?years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.     

Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density

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.     

A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species

Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.     

Suitability of immobilized metal affinity chromatography for protein purification from canola

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.     

Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level

Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.     

Metal binding by citrus dehydrin with histidine-rich domains

Dehydrins are hydrophilic proteins that are responsive to osmotic stress, such as drought, cold, and salinity in plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, their functions are not fully understood. Citrus dehydrin, which accumulates mainly in response to cold stress, enhances cold tolerance in transgenic tobacco by reducing lipid peroxidation. It has been demonstrated that citrus dehydrin scavenges hydroxyl radicals. In this study, the metal binding of citrus dehydrin is reported and the specific domain responsible is identified. The metal binding property of citrus dehydrin was tested using immobilized metal ion affinity chromatography (IMAC). Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ bound to citrus dehydrin, but Mg2+, Ca2+, and Mn2+ did not. Among the bound metals, the highest affinity was detected for Cu(2+)-dehydrin binding, which showed a dissociation constant of 1.6 microM. Citrus dehydrin was able to bind up to 16 Cu2+ ions. IMAC indicated that His residues contributed to Cu(2+)-dehydrin binding. The amino acid sequence of CuCOR15 was divided into five domains, of which domain 1 bound Cu2+ most strongly. One portion of domain 1, HKGEHHSGDHH, was the core sequence for the binding. These results suggest that citrus dehydrin binds metals using a specific sequence containing His. Since citrus dehydrin is a radical-scavenging protein, it may reduce metal toxicity in plant cells under water-stressed conditions.     

Simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) products and human saliva by gas chromatography-mass spectrometry

This paper reports on the selectivity behaviour of tryptic peptides on a Cu(2+)-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10-75 mM SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 mM) was characterised by MALDI-TOF. When using SDS at 75 mM, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.