Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM^®) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM^® Disk with epoxy chemistry. After this, the immobilized CIM^® Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM^® Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap^® metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.     

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological‐based specificity of the biomolecule–ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye‐affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α‐chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N‐hexyl pendant chain, with a ligand density of 1.8 × 10^?2 mmol of dye/g of chromatographic support, to isolate lysozyme, α‐chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α‐chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography

Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50–60% range for affinity resins, and in the 80–85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.     

Formation of high-capacity protein-adsorbing membranes through simple adsorption of poly(acrylic acid)-containing films at low pH

Layer-by-layer polyelectrolyte adsorption is a simple, convenient method for introducing ion-exchange sites in porous membranes. This study demonstrates that adsorption of poly(acrylic acid) (PAA)-containing films at pH 3 rather than pH 5 increases the protein-binding capacity of such polyelectrolyte-modified membranes 3-6-fold. The low adsorption pH generates a high density of -COOH groups that function as either ion-exchange sites or points for covalent immobilization of metal-ion complexes that selectively bind tagged proteins. When functionalized with nitrilotriacetate (NTA)-Ni(2+) complexes, membranes containing PAA/polyethylenimine (PEI)/PAA films bind 93 mg of histidine(6)-tagged (His-tagged) ubiquitin per cm(3) of membrane. Additionally these membranes isolate His-tagged COP9 signalosome complex subunit 8 from cell extracts and show >90% recovery of His-tagged ubiquitin. Although modification with polyelectrolyte films occurs by simply passing polyelectrolyte solutions through the membrane for as little as 5 min, with low-pH deposition the protein binding capacities of such membranes are as high as for membranes modified with polymer brushes and 2-3-fold higher than for commercially available immobilized metal affinity chromatography (IMAC) resins. Moreover, the buffer permeabilities of polyelectrolyte-modified membranes that bind His-tagged protein are ~30% of the corresponding permeabilities of unmodified membranes, so protein capture can occur rapidly with low-pressure drops. Even at a solution linear velocity of 570 cm/h, membranes modified with PAA/PEI/PAA exhibit a lysozyme dynamic binding capacity (capacity at 10% breakthrough) of ~40 mg/cm(3). Preliminary studies suggest that these membranes are stable under depyrogenation conditions (1 M NaOH).     

3,3′‐Diamino‐N‐methyldipropylamine as a versatile affinity ligand

Currently, in biomedicine and biotechnology fields, there is a growing need to develop and produce biomolecules with a high degree of purity. To accomplish this goal, new purification methods are being developed looking for higher performance, efficiency, selectivity, and cost‐effectiveness. Affinity chromatography is considered one of the most highly selective methods for biomolecules purification. The purpose of this work is to explore a new type of a structurally simple ligand immobilized onto an agarose matrix to be used in affinity chromatography. The ligand in this study, 3,3′‐diamino‐N‐methyldipropylamine has shown low toxicity and low cost of preparation. Moreover, the ability of the ligand to be used in affinity chromatography to purify proteins and nucleic acids was verified. An increasing sodium chloride gradient, using salt concentrations up to 500 mM, was suitable to accomplish the purification of these biomolecules, meaning that the new support allows the recovery of target biomolecules under mild conditions. Thus, the 3,3′‐diamino‐N‐methyldipropylamine ligand is shown to be a useful and versatile tool in chromatographic experiments, with very good results either for proteins or supercoiled plasmid isoform purification.     

Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM‐Asp) as chelating ligand and Ni²⁺ as center ion on the base of monodispersed, 3.0 µm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min^?1 by using the synthesized Ni²⁺‐CM‐Asp‐P_GMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni²⁺‐CM‐Asp‐P_GMA/EDMA column was further investigated for the rapid separation and purification of copper‐zinc superoxide dismutase (Cu,Zn‐SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Interactions between an amphipathic di‐histidine peptide and a metal affinity chromatographic resin derived from a bis(tacn)butane chelating ligand

The interactive behavior of an amphipathic peptide with the Cu²⁺, Ni²⁺, and Zn²⁺ complexes of 1,4‐bis(triazacyclonon‐1‐yl)butane), bis(tacn)^but, immobilized onto Sepharose CL‐4B, has been investigated. The effects of incubation time, as well as the incubation buffer pH and ionic strength, have been examined. The binding data have been interrogated using Langmuir, Langmuir‐Freundlich, bi‐Langmuir, and Temkin isothermal models and Scatchard plots. These results confirm that this amphipathic peptide binds with relatively high capacities to the immobilized Cu²⁺‐ and Ni²⁺‐1,4‐bis(triazacyclonon‐1‐yl)butane)‐Sepharose CL‐4B sorbents via at least two discrete sites. However, the corresponding immobilized Zn²⁺‐sorbent had low binding capacity. Moreover, the magnitude of the binding capacities of these sorbents was dependent on the pH and ionic strength of the incubation buffer. These results are relevant to the isolation of E. coli expressed recombinant proteins that incorporate this and related amphipathic peptide tags, containing two or more histidine residues, located at the N‐ or C‐terminus of the recombinant protein, and the co‐purification of low abundance host cell proteins of diverse structure, by immobilized metal ion affinity chromatographic methods.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Ethylenediamine-Derived Chromatographic Ligand to Separate BSA,Lysozyme, and RNase A

Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.

     

Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe³⁺, Ga³⁺, and UO₂ ²⁺ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe³⁺ and Ga³⁺ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga³⁺ and Fe³⁺ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.     

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates

The aim of this work was to devise a one‐step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild‐type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)–IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K_D of 4.53 × 10⁻⁷ m was obtained from batch isotherm measurements. The combination of tailor‐made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one‐step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial‐Zn(II) and EPI‐30–IDA–Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A‐Sepharose CL‐4B. This MAb preparation revealed on SDS–PAGE two protein bands with M_r of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Antifungal evaluation of cholic acid and its derivatives on Candida albicans by microcalorimetry and chemometrics

Multi-hydroxyl amines including tris(hydroxymethyl)aminomethane (Tris), serinol and ethanolamine were selected as weak affinity ligands using a rapid screening by quartz crystal microbalance (QCM) biosensor. Based on the specific recognition between the ligands and two proteins, lysozyme (LZM) and cytochrome c (Cyt c), a weak affinity chromatography method was developed for specific separation of the two proteins. The frontal analysis results showed that the apparent dissociation constants (K_D) of ligand–protein complexes were all in the order of weak affinity (10⁻⁴ M). By weak affinity columns modified with the three multi-hydroxyl amines individually, LZM and Cyt c were baseline separated as retarded peaks from non-specific protein and each other in a single cycle of loading and eluting. Moreover, the Tris-modified column typically showed the satisfactory repeatability and stability as a new type of weak affinity columns. The present strategy composed of QCM selecting and affinity chromatography separating was promising to extend the variety of weak affinity ligands and develop inexpensive specific affinity methods for separation and purification of multiple proteins on one single column.     

High-Speed Purification of Recombinant Interleukin-11 by IMAC with Rigid Biporous Beads

Rigid biporous beads were prepared and modified by iminodiacetic acid (IDA) for application in immobilized metal affinity chromatography of proteins. The retention behavior of four model proteins on the metal chelate columns loaded with copper (II) and nickel (II) ions were studied. The separation of the four proteins by the Ni-IDA column at 40 cm.min⁻¹ was realized within 2 min. His6-interluekin-11 (His6-IL-11) was also purified by the Ni-IDA column at 40 cm.min⁻¹. The collected His6-IL-11 fraction showed a purity of about 80%. The results indicate that the IMAC with the biporous medium is promising for high-speed protein purification.     

Synthesis and characterization of D-Alanyl-D-Alanine-Agarose

A new affinity adsorbent, using D-alanyl-D-alanine as ligand, has been prepared. The dipeptide immobilized on Activated CH-Sepharose 4B (D-Ala-D-Ala-AGA) bioselectively binds the glycopeptide antibiotics teicoplanin, vancomycin, ristocetin A (vancomycinlike group of antibiotics) while it does not bind other antibiotics equally active on cell wall biosynthesis but with different target sites, such as penicillin G, cephalosporin C, gardimycin, and bacitracin. Teicoplanin, vancomycin, and ristocetin A have similar binding characteristics for the immobilized dipeptide, as indicated by equilibrium binding experiments. The affinity constants of the three antibiotics for D-Ala-D-Ala-AGA is of the same order of magnitude (10⁵ L mol^-1) and the number of effective binding sites is similar for each antibiotic (6–7 μEq/mL of gel). The adsorption is biospecific as no binding has been observed to immobilized L-alanyl-L -alanine. D-Ala-D-Ala-AGA has been successfully used to purify teicoplanin from mixtures of different complexity and for concomitant extraction and purification from fermentation liquors by both batch adsorption and column chromatography. The antibiotic can be recovered from the resin in high yields by elution at pH 11.     

Poly(α-amino acid)-immobilized polymer adsorbents for optical resolution. I. Synthesis,characterization, and evaluation of poly(L-glutamic acid) derivatives-immobilized polymer adsorbents

As a novel polymer adsorbent for optical resolution, cross-linked polystyrene gel incorporating poly(α-amino acids) was synthesized. The helicity of the incorporated poly(γ-benzyl L -glutamate) (PBLG) was demonstrated by Fourier-transform infrared spectroscopy. The immobilized PBLG ( I ) was converted to poly(L -glutamic acid) ( II ) and poly(N⁵-benzyl-L -glutamine) ( III ). The ability of I - III to resolve DL-mandelic acid was evaluated by liquid chromatography using toluene/dioxane as an eluent. Of the three resins, III resolves the recemate most effectively. In order to clarify the mechanism of chiral recognition, poly(N⁵-benzyl-D -glutamine) and poly(N⁴-benzyl-L -asparagine), with opposite helicity, was incorporated. In Contrast to III , these adsorbents demonstrated affinity for the L isomer. This result strongly indicates that the helical structure of the immobilized poly(α-amino acids) is essential for chiral recognition.     

Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn<Superscript>2+</Superscript> and -Co<Superscript>2+</Superscript> Affinity Chromatography

Coagulation factor VIII (FVIII) is a glycoprotein that plays a crucial role in the clotting cascade. Replacement therapies with recombinant and plasma-derived concentrates of FVIII are used for treatment of hemophilia A. We have previously purified the human plasma FVIII by immobilized metal affinity chromatography (IMAC) using Cu²⁺ as the metal ligand. In this work we report the purification of FVIII using Zn²⁺ and Co²⁺, two metal ions that bind proteins more weakly. Human plasma was directly applied to the anion-exchange ANX Sepharose FF column and the eluate was used as starting material for the studies in IMAC columns. Using imidazole as desorbing agent, FVIII was recovered with 65% activity in the IMAC-Zn²⁺ column and with 74% activity in the IMAC-Co²⁺ column. Purification factors were 4 and 9, respectively. Using a pH gradient, FVIII was eluted at pH 5.0 with 17% activity in the IMAC-Zn²⁺ and 77% activity in the IMAC-Co²⁺. Vitamin K-dependent proteins, a family of proteins that includes Prothrombin and coagulation factor IX, coeluted with FVIII in the ANX Sepharose FF column and were recovered with the unbound proteins on both IMAC columns. Therefore, Co²⁺ and Zn²⁺ columns were as effective as the Cu²⁺ column in separating FVIII from vitamin K-dependent proteins. Finally, we have shown that FVIII remained complexed with the von Willebrand factor.     

Rapid and efficient one‐step purification of a serralysin family protease by using a p‐aminobenzamidine‐modified affinity medium

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p‐aminobenzamidine‐modified affinity chromatography. The affinity medium was synthesized by using p‐aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant K _d and theoretical maximum adsorption Q _max of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high‐performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.     

A fullerene C<Subscript>60</Subscript>-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.     

Design of a ruthenium(III) immobilized affinity material based on a β‐cyclodextrin‐functionalized poly(glycidyl methacrylate‐ethylene dimethacrylate) monolith for the enrichment of hippuric acid

Immobilized metal affinity chromatography has drawn great attention as a widespread separation and purification approach. In this work, ruthenium was firstly introduced into the preparation of immobilized metal affinity chromatography considering its affinity to N,O‐donor ligands. A β‐cyclodextrin‐functionalized poly(glycidyl methacrylate‐ethylene dimethacrylate) monolith was designed and employed as the supporting material in immobilized metal affinity chromatography. Thiosemicarbazide was introduced into the synthesis process, which not only acted as a bridge between β‐cyclodextrin and glycidyl methacrylate, but also chelated with ruthenium because of its mixed hard‐soft donor characteristics. The developed monolithic ruthenium(III)‐immobilized metal affinity chromatography column was utilized for the adsorption and separation of hippuric acid, a biological indicator of toluene exposure. To achieve high extraction capacity, the parameters affecting the extraction efficiency were investigated with an orthogonal experiment design, L9 (3⁴). Under the optimized conditions, the enrichment factor of hippuric acid was 16.7‐fold. The method reproducibility was investigated in terms of intra‐ and interday precisions with relative standard deviations lower than 8.7 and 9.5%, respectively. In addition, ruthenium(III)‐immobilized metal affinity chromatography material could be used for up to 80 extractions without an apparent change in extraction recovery.