Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu²⁺ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH₄Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH₄Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH₄Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97–100%).     

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.     

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.     

Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.     

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu²⁺/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.     

Anti-free-radical properties of the peptide fractions isolated from string bean by immobilized metal ion affinity chromatography

In this study, peptides and proteins extracted from string bean with 1% TCA were separated by chromatography on columns with copper ions immobilized through IDA and o-phosphoserine OPS. Protein and peptide concentrations and the anti-free-radical properties of the isolated fractions were determined. Identification was obtained using mass spectrometry. The anti-free- radical activity of all analyzed samples was determined using the DPPH test, and was found to depend on reaction time, choice of chelating agent and the order in which the fractions were eluted from the column.     

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.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Design of affinity tags for one-step protein purification from immobilized zinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.

Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.     

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.     

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q_max) of Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate for the GFP was 1,42,860 FU ml⁻¹ and 18,000 FU ml⁻¹, respectively. The expanded bed column chromatography using Ni²⁺ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni²⁺ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.     

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 Zn²⁺ 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.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.