Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers

Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments.     

重组人Fab金属螯合层析法纯化条件的研究

在重组人Fab(rh Fab)表达载体的羧基端插入六个组氨酸, 使其对金属螯合层析介质产生特异性吸附, 可用金属螯合亲和层析法进行分离纯化. 采用自制金属(铜、锌金属离子)螯合层析介质, 以pH和咪唑两种洗脱方法,对rh Fab段的纯化效果进行了探讨. 结果显示: 铜离子螯合层析介质比锌离子螯合层析介质对rh Fab的亲和能力更强; pH洗脱方法的重复性优于咪唑法; 金属铜离子螯合层析法对rh Fab进行一步纯化可得到纯度大于95%的rh Fab产品.     

Deconvoluting the Cu2+ binding modes of full-length prion protein

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.     

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.     

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.     

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.     

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.     

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.     

Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.     

Effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor (Cys17Ser) in aqueous two-phase systems containing chelated metal ions

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.     

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.     

Separation of human Fab fragments on negative mode Ni(II)-TREN-agarose chromatography

We evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) with nickel ion complexed with Tris(2-aminoethyl)amine (TREN) immobilized on agarose gel for purification of human Fab fragments by negative chromatography. Efficient purification of Fab fragments from digested human IgG (immunoglobulin G) (106.4% purity) was accomplished in Tris-HCl buffer at pH 7.5 without NaCl (based on total protein concentration and radial immunodiffusion of human Fab). A technological application of Ni(II)-TREN-agarose using non transgenic soybean protein extract spiked with human Fab fragments as feedstream was also studied. Experiments using Tris-HCl at pH 7.0 as loading buffer allowed the adsorption of almost all of the soybean proteins. Sixty-six percent of the loaded human Fab fragments were recovered in the flowthrough and washing fractions with about 90% purity. These results demonstrate that Ni(II)-TREN-agarose is a potential adsorbent for recombinant Fab fragment purification.     

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Nickel(II) complexes of the multihistidine peptide fragments of human prion protein

Nickel(II) complexes of the peptide fragments of human prion protein containing histidyl residues both inside and outside the octarepeat domain have been studied by the combined application of potentiometric, UV-visible and circular dichroism spectroscopic methods. The imidazole-N donor atoms of histidyl residues are the exclusive metal binding sites below pH 7.5, but the formation of stable macrochelates was characteristic only for the peptide HuPrP(76-114) containing four histidyl residues. Yellow colored square planar complexes were obtained above pH 7.5-8 with the cooperative deprotonation of three amide nitrogens in the [N_im,N⁻,N⁻,N⁻] coordination mode. It was found that the peptides can bind as many nickel(II) ions as the number of independent histidyl residues. All data supported that the complex formation processes of nickel(II) are very similar to those of copper(II), but with a significantly reduced stability for nickel(II), which shifts the complex formation reactions into the slightly alkaline pH range. The formation of coordination isomers was characteristic of the mononuclear complexes with a significant preference for the nickel(II) binding at the histidyl sites outside the octarepeat domain. The results obtained for the two-histidine fragments of the protein, HuPrP(91-115), HuPrP(76-114)H85A and HuPrP(84-114)H96A, made it possible to compare the binding ability of the His96 and His111 sites. These data reveal a significant difference in the nickel(II) and copper(II) binding sites of the peptides: His96 was found to predominate almost completely for nickel(II) ions, while the opposite order, but with comparable concentrations, was reported for copper(II).     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.     

Steroid-binding specificity of human sex hormone-binding globulin is influenced by occupancy of a zinc-binding site

One calcium-binding site (site I) and a second poorly defined metal-binding site (site II) have been observed previously within the amino-terminal laminin G-like domain (G domain) of human sex hormone-binding globulin (SHBG). By soaking crystals of this structure in 2.5 mm ZnCl(2), site II and a new metal-binding site (site III) were found to bind Zn(2+). Site II is located close to the steroid-binding site, and Zn(2+) is coordinated by the side chains of His(83) and His(136) and the carboxylate group of Asp(65). In this site, Zn(2+) prevents Asp(65) from interacting with the steroid 17beta-hydroxy group and alters the conformations of His(83) and His(136), as well as a disordered region over the steroid-binding site. Site III is formed by the side chains of His(101) and the carboxylate group of Asp(117), and the distance between them (2.7 A) is increased to 3.7 A in the presence of Zn(2+). The affinity of SHBG for estradiol is reduced in the presence of 0. 1-1 mm Zn(2+), whereas its affinity for androgens is unchanged, and chemically-related metal ions (Cd(2+) and Hg(2+)) have similar but less pronounced effects. This is not observed when Zn(2+) coordination at site II is modified by substituting Gln for His(136). An alteration in the steroid-binding specificity of human SHBG by Zn(2+) occupancy of site II may be relevant in male reproductive tissues where zinc concentrations are very high.