Immobilized Metal Affinity Chromatography Adsorbent Research Articles

Dual column approach for the purification of zinc finger proteins by immobilized metal affinity chromatography

Abstract The feasibility of using zinc fingers as the affinity tag for the purification of recombinant proteins with immobilized metal affinity chromatography (IMAC) was investigated. It was shown that upon the release of pre-existing metal ions from the model zinc-finger fusion protein by dialysis with buffer containing 500 mM NaCl, the recovery of the zinc-finger fusion protein was increased from 47% to 87%. The purity of the zinc-finger fusion protein was further improved with a pre-precipitation step at pH 5.0. The adsorption capacities for the zinc-finger fusion protein were in the order of Ni(II) > Cu(II) > Zn (II) > Co(II). Based on the low affinity of Zn(II)-loaded IMAC adsorbent for the model protein, a dual column process with Zn(II)-IMAC as the negative column for the removal of unwanted proteins and Ni(II)-IMAC as the positive column for the adsorption of the zinc-finger fusion protein was proposed, giving a purity of 98%. Purification of the zinc-finger fusion protein under denaturing conditions was also demonstrated. The results of this study suggest that it is possible to purify zinc finger proteins with IMAC without the need for external affinity tags, which is particularly useful for the purification of zinc finger proteins for structural analysis.

Novel bis(5-methyltetrazolium)amine ligand-bonded stationary phase with reduced leakage of metal ions in immobilized metal affinity chromatography of proteins.

Immobilized metal affinity chromatography (IMAC) has been widely used for the specific separation of biopolymers. However, leakage of metal ions from IMAC adsorbents is of concern in IMAC. In this study, we designed a novel tridenate bis(5-methyltetrazolium)amine (BMTA) to reduce the leakage of metal ions by improving the affinity to immobilized metal ions. The ligand was bonded onto silica via three-step reaction to prepare a high-performance IMAC stationary phase. The chromatographic behaviors of ribonuclease A, cytochrome c, and lysozyme on the Cu(II)-, Ni(II)-, and Zn(II)-chelated stationary phase were investigated with respect to pH effect and elution with an imidazole gradient. The retention times of these three proteins increased by increasing the pH of the mobile phase but decreased by increasing the concentration of the competitive displacer. The retaining strength of the three proteins on the chelated stationary phase were in the order Cu(II) > Ni(II) > Zn(II). The behavior of these three proteins was consistent with the properties of a typical IMAC. The BMTA ligand exhibited a much stronger affinity for Cu(II) and Ni(II) than iminodiacetic acid (IDA), which is often regarded as a standard tridentate IMAC ligand. Quantum mechanical calculations at the B3LYP/6-31G level were used to image the coordination mode of the protein-metal ions-BMTA complex. In addition, a fused histidine-tagged cecropin b-human epidermal growth factor (CB-EGF) from Escherichia coli crude extract was purified by the Ni(II)-chelated stationary phase, and the purity of the CB-EGF was determined to be at least 90%. These results suggest that the BMTA ligand may have potential applications in the preparation of therapeutics. Graphical Abstract A novel ligand of tridenate bis(5-methyltetrazolium)amine (BMTA) was designed to reduce the leakage of metal ions from the column in immobolized metal affinity chromatography (IMAC).

Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents

A systematic analysis on the adsorption of homo-oligomeric, poly(His)-tagged recombinant proteins on a hydroxyapatite-based immobilized metal affinity chromatography adsorbent was performed. Under non-denaturing conditions, the Langmuir–Freundlich isotherm model was found ideal for predicting the adsorption behavior of the model proteins. The dissociation constants, as low as 10 −9 M, decreased with the number of poly(His) tags, suggesting the presence of multi-point attachment. The maximum adsorption capacities, ranging from 79.1 nmol/g for the 88 kDa epimerase to 42.8 nmol/g for the 320 kDa racemase, were inversely proportional to the contact surface areas of the proteins. Under denaturing conditions, the Langmuir isotherm model fitted well with the experimental data. The maximum adsorption capacities for and the dissociation constants of the three model proteins were essentially identical, as the subunits of the model proteins were of similar dimensions and behaved similarly in the absence of complex tertiary or quaternary structure. Phosphate buffer at a concentration of 500 mM, pH 8.0 was found to be effective for the elution of the model proteins with a recovery yield more than twofold higher than that obtained with 500 mM imidazole. The results suggest the hydroxyapatite-based adsorbent is a promising alternative for large scale applications.

Immobilized-metal affinity chromatography adsorbent with paramagnetism and its application in purification of histidine-tagged proteins

A new method for synthesis of an Immobilized-Metal Affinity chromatography (IMAC) adsorbent with superparamagnetism (Fe 3O 4/SiO 2-GPTMS-Asp-Co) was reported in this paper. Fe 3O 4 nanoparticles were first modified by SiO 2 to form the core–shell Fe 3O 4/SiO 2 with superparamagnetism, the core–shell microspheres were then successively treated by 3-glycidoxypropyltrimethoxysilane (GPTMS), l-aspartic acid ( l-Asp) and 2-bromoacetic acid to form Fe 3O 4/SiO 2-GPTMS-Asp nanoparticles with tetradentate ligands. Finally, the IMAC adsorbent with superparamagnetism, Fe 3O 4/SiO 2-GPTMS-Asp-Co, was finally obtained by the coordination of Co 2+ with the resulting nanoparticles. The intermediates and product obtained from the process mentioned above were characterized by TEM, SEM, XRD, XPS, FT-IR, TGA, AAS, EDS, element analysis and magnetic hysteresis loop. Fe 3O 4/SiO 2-GPTMS-Asp-Co nanoparticles used as IMAC adsorbent to separate the recombinant 6-histidine-tagged gp41 protein were investigated. The result indicates that the IMAC adsorbent we prepared has outstanding advantages in the separation of the 6-histidine-tagged proteins from the crude bacterial lysate, such as simple operation, high selectivity and capacity.

Adsorption behaviors of recombinant proteins on hydroxyapatite-based immobilized metal affinity chromatographic adsorbents

The equilibrium adsorption of three homo-oligomeric model recombinant proteins containing up to 8 poly(histidine) affinity tags on a hydroxyapatite-based immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The experimental data are well fitted with the three-parameter Langmuir–Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins. The maximum capacity and the binding affinity of the IMAC adsorbent for the model proteins are in principle dependent on the size and the number of affinity tags of proteins, respectively. The exceptionally high association constant of the octameric racemase, probably due to simultaneous multipoint attachment, make it difficult to elute racemase from the adsorbent. The adsorption isotherms under denaturing conditions are well fitted with the Langmuir model. Results of Scatchard analysis further suggest the homogeneous adsorption of the model protein subunits under denaturing conditions. The binding capacities and affinities of the adsorbent under denaturing conditions for the three unfolded protein subunits become essentially identical because the molecular size and number of poly(His) tags of the unfolded polypeptide chains of the three protein subunits are the same. The significant reduction in association constants under denaturing conditions suggests that high concentration of urea could interfere with the binding of proteins on the hydroxyapatite-based adsorbent.

A poly(2-hydroxyethyl methacrylate)-based immobilized metal affinity chromatography adsorbent for protein purification

Poly(HEMA) microbeads were prepared by suspension polymerization of 2-hydorxyethylmethacrylate and ethyleneglycoldimethacrylate (EGDMA). The water content, ligand density, and selectivity for poly(His)-tagged d-hydantoinase of the poly(HEMA)-based adsorbents were affected by the concentration of EDGMA used during polymerization. The Ni(II)-loaded poly(HEMA) adsorbent exhibited an adsorption capacity of 1.0 mg/g for poly(His)-tagged d-hydantoinase under optimal conditions with buffer containing 100–300 mM NaCl at pH 6.0. One-step purification protocol with the adsorbent gave a purity of at least 92%. The adsorption capacity of adsorbent declined by 54% after 7 cycles, due to the leaching of Ni(II) from the adsorbent. However, upon regeneration the adsorption capacity can be restored. Given the ease of preparation and the chemical and microbial resistance, the poly(HEMA)-based IMAC adsorbent could be a promising substitute for the polysaccharide-based IMAC adsorbents.

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.

Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents

The equilibrium adsorption of four homo-oligomeric recombinant proteins containing up to eight poly(histidine) affinity tags on a silica-based Cu(II)-IDA immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The equilibrium adsorption data are well fitted with the three-parameter Langmuir–Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins and the degree of cooperativity seems to increase with the number of poly(His) tags. This phenomenon is also supported by the concave-downward curves in the Scatchard analysis. The maximum capacities the IMAC adsorbent for the model proteins, declining from 31.79 nmol/g gel for 88 kDa epimerase to 21.50 nmol/g gel for 320 kDa racemase, are in principle affected by the size of the molecule, and the binding strengths, expressed in term of association constants K a increasing from 1.47 μM −1 for the dimeric epimerase to 50.01 μM −1 for the octameric racemase, are dictated by the number of affinity tags. Nevertheless, some structure-specific interactions observed among native N-carbamoylase molecules may promote multilayer adsorption and thus lead to a much higher adsorption capacity, 715 nmol/g gel and a lower compounded binding affinity, 0.42 μM −1. The observed positive cooperativity for the model proteins and the structure-specific interactions among N-carbamoylase molecules are eliminated under denaturing conditions, resulting in a homogeneous adsorption well fitted by the Langmuir isotherm model.

Macroporous chitosan layer coated on non-porous silica gel as a support for metal chelate affinity chromatographic adsorbent

A new immobilized metal affinity chromatography (IMAC) matrix was prepared by coordinating Cu 2+ with cross-linked chitosan coated on non-porous silica gel (Cu–CTS–SiO 2). Macroporous structure could be formed on the coated layer by imprinting polyethylene glycol (PEG) in chitosan film. The surface morphology changes on Cu–CTS–SiO 2 bead prepared in different condition were confirmed by scanning electron microscopy (SEM). Effects of chitosan and PEG content in coating solution, the molecular mass of PEG on the surface macropore formation and adsorption capacity of bovine serum albumin (BSA) were investigated. Results indicated that coating solution with 2% chitosan and 10% PEG 20000 was optimal. Batch experiments were also conducted for elucidating the optimal pH, the adsorption isotherm and adsorption kinetics of BSA. Adsorption isotherm of trypsin on the same adsorbent was also performed. Results showed that the support itself had low non-specific interaction with both BSA and trypsin. The maximum adsorption capacity for BSA and trypsin on the prepared IMAC adsorbent could reach 192 mg and 5000 IU, respectively calculated by every gram of chitosan. The binding and eluting condition for BSA were tested on column filled with the adsorbent. Crude BSA sample could be purified on the IMAC column.

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 Cu 2+/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.

Procion Brown MX-5BR attached and Lewis metals ion-immobilized poly(hydroxyethyl methacrylate)/chitosan IPNs membranes: Their lysozyme adsorption equilibria and kinetics characterization

Interpenetration networks (IPNs) in membrane form were synthesized from 2-hydroxyethyl methacrylate (HEMA) and chitosan (pHEMA/chitosan) via UV-initiated photo-polymerization in the presence of an initiator α, α’-azoisobutyronitrile. Procion Brown MX-5BR (PB MX-5BR) was covalently attached onto IPNs membrane as a metal chelating dye-ligand. Two different Lewis metal ions (Fe(III) or Cu(II)) were immobilized onto the dye-ligand for utilization in the immobilized metal affinity chromatography (IMAC). The binding characteristics of a model protein (lysozyme) to IMAC adsorbents and selectivity of immobilized metal ions (Fe(III) and Cu(II)) to the lysozyme have been investigated from aqueous solution using the dye-ligand-attached IPNs membrane as a control system. The experimental data was analysed using the two adsorption kinetic models the pseudo-first-order and pseudo-second order to determine the best-fit equation for the adsorption of lysozyme onto dye-ligand and IMAC adsorbents. The second-order equation for the adsorption of lysozyme on the dye-ligand, dye-ligand–Fe(III) and dye-ligand–Cu(II) membrane systems is the most appropriate equation to predict the adsorption capacity for all the tested adsorbents. The reversible lysozyme adsorption on the dye-ligand and IMAC adsorbents obeyed the Temkin isotherm. The lysozyme adsorption capacities of the dye-ligand, dye-ligand–Fe(III) and dye-ligand–Cu(II) immobilized IPNs membranes were 79.1, 147.4, and 128.2 mg ml −1 , respectively. The adsorption of the lysozyme on the pHEMA/chitosan membrane plain was about 8.3 mg ml −1 .