Immobilized Metal Affinity Chromatography Column Research Articles

A simple and high efficiency purification of His-tagged turnip yellow mosaic virus-like particle (TYMV-VLP) by nickel ion affinity precipitation

Virus-like particles (VLPs) is one of the most favourable subjects of study, especially in the field of nanobiotechnology and vaccine development because they possess good immunogenicity and self-adjuvant properties. Conventionally, VLPs can be tagged and purified using affinity chromatography or density gradient ultracentrifugation which is costly and time-consuming. Turnip yellow mosaic virus (TYMV) is a plant virus, where expression of the viral coat protein (TYMVc) in Escherichia coli (E. coli) has been shown to form VLP. In this study, we report a non-chromatographic method for VLP purification using C-terminally His-tagged TYMVc (TYMVcHis6) as a protein model. Firstly, the TYMVcHis6 was cloned and expressed in E. coli. Upon clarification of cell lysate, nickel (II) chloride [NiCl2; 15 µM or equivalent to 0.0000194% (w/v)] was added to precipitate TYMVcHis6. Following centrifugation, the pellet was resuspended in buffer containing 1 mM EDTA to chelate Ni2+, which is then removed via dialysis. A total of 50% of TYMVcHis6 was successfully recovered with purity above 0.90. Later, the purified TYMVcHis6 was analysed with sucrose density ultracentrifugation, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to confirm VLP formation, which is comparable to TYMVcHis6 purified using the standard immobilized metal affinity chromatography (IMAC) column. As the current method omitted the need for IMAC column and beads while significantly reducing the time needed for column washing, nickel affinity precipitation represents a novel method for the purification of VLPs displaying poly-histidine tags (His-tags).

Improved Catalytic Reaction of Biotemplated Palladium Nanoparticles through Immobilized Metal Affinity Purification

To investigate the effect purification plays on nanoparticle (NP) synthesis and catalytic activity, three copies of Pd4 (TSNAVHPTLRHL) fused to the N-terminus of Green Fluorescent Protein (GFP) was produced recombinantly and its characteristics pre and post purification was assessed. An E. coli expression system was employed, and purification was performed with Immobilized Metal Affinity Column (IMAC). Transmission electron microscopy (TEM) was utilized to examine the morphology of NPs synthesized with an enriched protein sample and ImageJ was used to determine the average size to be 2.44 nm. The turnover frequency of fabricated NP from the purified protein was analyzed by a model Suzuki-Miyaura coupling reactions and determined to be 33,000 hr-1. This value is three times higher than the turnover frequency when crude lysate containing (Pd4)3-GFP was used during NP synthesis. This result shows that enrichment enhanced the catalytic activity of NP.

ISOLATION AND PURIFICATION OF AVIDIN FROM EGG WHITE BY IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY

Lysozyme and avidin were initially separated from egg white by cation exchange chromatography (Doulite C-464). Avidin was then isolated from lysozyme in pure form by immobilized metal affinity chromatography column (1.5 x 10cm) loaded with copper ions. The column was equilibrated with 0.02M phosphate buffer pH 7.7, containing 0.5M NaCl. Protein fraction obtained from Doulite column was applied on IMAC column, followed by washing with the starting buffer and eluting with pH gradient with 0.1M Acetic acid. Two peaks were obtained, the first peak represents the avidin, while the second peak represents the lysozyme as tested by SDS-PAGA and HABA assay. The purity of avidin was increased to 75% by the IMAC process.

Profiling protein interactions by purification with capillary monolithic affinity column in combination with label-free quantitative proteomics

An approach for profiling protein-protein interactions by using affinity purification with capillary monolithic immobilized metal affinity chromatography column (cm-IMAC) in combination with label free quantitative proteomics was described in the present work. The cm-IMAC columns were prepared in a single step by copolymerization of the function monomer, namely (S)-2,2′-((1-carboxy-5-(pent‑4-enamido)pentyl)azanediyl)diacetic acid which provide a nitrilotriacetate (NTA) moiety to form chelated complexation with Ni (II) ions, inside the fused silica capillaries. The His6-tagged bait protein can be easily immobilized on the cm-IMAC columns through the formation of chelating complexation with the NTA-Ni (II) functional groups of the matrix. The cm-IMAC columns were used to explore protein-protein interactions (PPIs) on a proteomic scale when combined with label-free proteomics. A known interaction pair of proteins, namely NDP52 (amino acid sequence 10–126) and NAP1 (33–75) as well as Bcl-2 family proteins were used for proof of concept. New interactors of Bcl-XL were identified and validated by co-immunoprecipitation.

Recombinant turnip yellow mosaic virus coat protein as a potential nanocarrier.

To display a short peptide (GSRSHHHHHH) at the C-terminal end of turnip yellow mosaic virus coat protein (TYMVc) and to study its assembly into virus-like particles (TYMVcHis6 VLPs). In this study, recombinant TYMVcHis6 expressed in Escherichia coli self-assembled into VLPs of approximately 30-32nm. SDS-PAGE and Western blot analysis of protein fractions from the immobilized metal affinity chromatography (IMAC) showed that TYMVcHis6 VLPs interacted strongly with nickel ligands in IMAC column, suggesting that the fusion peptide is protruding out from the surface of VLPs. These VLPs are highly stable over a wide pH range from 3·0 to 11·0 at different temperatures. At pH 11·0, specifically, the VLPs remained intact up to 75°C. Additionally, the disassembly and reassembly of TYMVcHis6 VLPs were studied in vitro. Dynamic light scattering and transmission electron microscopy analysis revealed that TYMVcHis6 VLPs were dissociated by 7moll-1 urea and 2moll-1 guanidine hydrochloride (GdnHCl) without impairing their reassembly property. A 10-residue peptide was successfully displayed on the surface of TYMVcHis6 VLPs. This chimera demonstrated high stability under extreme thermal conditions with varying pH and was able to dissociate and reassociate into VLPs by chemical denaturants. This is the first C-terminally modified TYMVc produced in E. coli. The C-terminal tail which is exposed on the surface can be exploited as a useful site to display multiple copies of functional ligands. The ability of the chimeric VLPs to self-assemble after undergo chemical denaturation indicates its potential role to serve as a nanocarrier for use in targeted drug delivery.

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

Using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) a new series of alternative metal binding-5 (amb5) heptapeptides that provide various combinations of His, Cys and Asp as potential metal binding sites are compared for their relative formation of complexes with the metal ions Co(II), Ni(II) and Zn(II). These amb5 peptides have the general primary structure acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where Aa1,2,6,7 are the four amino acids that include the His, Cys, and/or Asp potential metal binding sites. The study includes two polyhistidine heptapeptides; the His-tag and His-tag with Pro4, because they represent the widely applied His affinity tag used in expression cloning and purification of recombinant proteins with immobilized metal affinity chromatography (IMAC). We compare these amb5 heptapeptides for their formation of the amb5+M(II) and amb5+M(II)+NTA complexes, where M = Co, Ni, or Zn, and NTA is nitrilotriacetic acid as a model for the binding of amb5 in a IMAC column. Overall, the results show that the inclusion of Asp at positions 1, 2, and 7; i.e., ac-Asp1-Asp2-Gly3-Pro4-Tyr5-Aa6-Asp7, generally favors the formation of amb5+Ni(II) and amb5+Ni(II)+NTA complexes, while the inclusion of Cys at positions 2 and 6; i.e., ac-Aa1-Cys2-Gly3-Pro4-Tyr5-Aa6-Cys7, with Aa1 = His, Cys or Asp, favors the formation of amb5+Zn(II) complexes. The comparison with the His-tag and competition between Ni(II) and Zn(II) for forming the amb5+M(II)+NTA complexes, showed the structure ac-Asp1-Asp2-Gly3-Pro4-Tyr5-His6-Aa7, with Aa7 = Cys or Asp, were the most selective for Ni(II) and forming the amb5+Ni(II)+NTA complex. Collision-induced dissociation of the single negatively-charged [amb5+Ni(II)+NTA]− shows it competitively dissociates into [amb5−3H+Ni(II)]− or [NTA−3H+Ni(II)]− while the doubly-charged [amb5+Ni(II)+NTA]2− complex dissociates into [NTA−3H+Ni(II)]− and [amb5J−H]−, indicating these complexes are realistic mimics for the complex inside an IMAC column.

Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate

In an effort to control and eventually eliminate malaria, the development of transmission-blocking vaccines has long been sought. However, few antigens have been evaluated in clinical trials, often due to limitations in the expression and purification of the antigen in sufficient yield and quality. Pfs230, a surface antigen of gametocytes, has recently advanced to clinical evaluation as a conjugate vaccine using the Pseudomonas aeruginosa exoprotein A carrier protein. Here we continue to build upon prior work of developing a Pfs230 candidate in the baculovirus system, Pfs230C1 (aa 443–731), through systematic process development efforts to improve yield and purity.Various insect cells including High Five, Sf9 and Super Sf9 were first evaluated for quality and quantity of antigen, along with three insect cell media. In the selection of Sf9 cells, an intact Pfs230C1 was expressed and harvested at 48 h for downstream development. A downstream process, utilizing immobilized metal affinity column (IMAC), followed by ion exchange (IEX) membranes (Mustang S) and finally IEX chromatography (DEAE) yielded a pure Pfs230C1 protein. The complete process was repeated three times at the 20 L scale.To support the eventual chemistry manufacturing and controls (CMC) of Pfs230C1, analytical tools, including monoclonal antibodies, were developed to characterize the identity, integrity, and purity of Pfs230C1. These analytical tools, taken in combination with the optimized process, were implemented with Current Good Manufacturing Practices (cGMP) in mind with the ultimate objective of Phase I clinical trials.

Size of protein is a major factor that affects retention on preparative IMAC columns

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6 × histidine, (His)6‐tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow‐rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine‐containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow‐through (output). Data obtained suggest that regardless of the flow‐rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium‐sized proteins (~50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.Support or Funding InformationWelch Foundation Grant # AN‐0008 SFASU CoSM SURE 2018This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and purification of thioredoxin-his6-ZmDREB2.7 fusion protein in Escherichia coli for raising antibodies

Dehydration-responsive element-binding (DREB) proteins play a critical role in the plant’s drought-tolerance mechanism despite their presence in minor amounts in the cell. In this study, a maize-derived transcription factor protein, ZmDREB2.7, was overexpressed in the Escherichia coli strain Rosetta 1. The interested gene conjugating with the thioredoxin gene (TrxA) and his6 tag in the pET-32a vector encoded a 55.7 kDa fusion protein. The optimum condition for inducing the thioredoxin-his6-ZmDREB2.7 expression was five hours of induction with 0.05 mM IPTG at 300C. The Tris-HCl 20 mM pH 8.0 lysis buffer was harnessed to extract the recombinant protein for the purification process. Using the immobilized-metal affinity chromatography column, the recombinant protein was purified and then injected into rabbits. The antisera containing polyclonal antibodies (pAbs) could specifically recognize the ZmDREB2.7 fusion protein. This study represents updated data on the bacterial expression of the recombinant ZmDREB2.7 protein and the production of anti-ZmDREB2.7 pAbs.

Study on the affinity characteristics of proteins on the immobilized metal affinity chromatography column

Study on affinity characteristic paramaters (KPML & ΛPML) of proteins on the iminodisuccinic acid metal chelate copper column by frontal chromatography.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the μCols compared with conventional columns for similar protein yields.

Size of Protein is a Major Factor that Affects Retention on Preparative IMAC Columns.

Immobilized metal affinity chromatography (IMAC) is a specific high-capacity technique used in large-scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6× histidine, (His)6-tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow-rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine-containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow-through (output). Data obtained suggest that regardless of the flow-rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium-sized proteins (~ 50kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.

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.

Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn2+ and -Co2+ 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.

Preparation and evaluation of multidentate ligand-bonded silica chromatographic medium

Iminodisuccinic acid (IDS), a green environment-friendly multidentate chelating agent, was used as a ligand to synthesize IDS-Silica stationary phase under the optimized conditions. The binding capacity of IDS on the stationary phase was measured by potentiometric titration. The chromatographic properties and metal chelating property on IDS-Silica column were investigated. Three standard protein mixtures were separated successfully with IDS-Silica column. The results showed that the IDS-Silica column displayed a typical cation exchange property. The binding characteristics of six different metal ions on IDS-Silica stationary phase were examined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The results validated that the variation of binding capacities of metal ions on the stationary phase were consistent with the chelating stability order of the IDS-Silica column for metal ions. Compared with the other silica columns bonded with the three different aminocarboxyl ligands, the bonding amounts of Cu2+ on IDS-Silica column was the largest, which indicated that IDS had stronger chelating ability for metal ions. This characteristic lays the foundation for IDS as a good chromatographic packing used in the field of immobilized metal affinity chromatography (IMAC), thus probably provides an effective solution to reduce the leaking problem of metal ion from IMAC column during protein elution with one competitive agent.

Opossum peptide that can neutralize rattlesnake venom is expressed in Escherichia coli.

An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre-incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life-saving therapy could be developed. To date, no recombinant therapies targeting cytotoxic envenomation have been reported. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:81-86, 2017.

Efficient In Vitro Refolding and Characterization of Major Histocompatibility Complex Class I-Related Chain Molecules A (MICA) and Natural Killer Group 2 Member D (NKG2D) Expressed in E. coli.

Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.

Camel Heavy Chain Polyclonal Antibody Raised Against Recombinant Murine Placental Growth Factor Expressed in Escherichia coli.

Angiogenesis, a neovascularization process, is the most important occurrence in developmental and pathological conditions. Key factors involve belonging to the vascular endothelial growth factor family. Recently, placental growth factor (PlGF) has been considered in medicine because of its pathological importance in solid tumor invasion and metastasis. Therefore, PlGF targeting plays a promising role in the hindrance of tumor progress. In this study, murine PlGF was cloned, expressed in an Escherichia coli system, and used for development of polyclonal camel antibody. Codon-optimized mouse PlGF (mPlGF) cDNA was synthesized and cloned in to pET-28a. The expression was performed in BL21 (DE3) E. coli strain and purified by immobilized metal affinity column chromatography. A camel was subcutaneously immunized six times over a one-week interval using purified protein. IgGs were purified by applying the serum on two sequential column affinity chromatography using proteins A and G. Then, anti-PlGF IgG was identified by Western bolt analysis and ELISA using commercial and expressed PlGF. Synthesized mPlGF was cloned successfully into the pET-28a expression vector, and the accuracy of construct was confirmed by map restriction analysis and sequencing. The expression was induced by 1 mM IPTG and confirmed by SDS-PAGE and Western blot using anti-His monoclonal antibody. The camel was immunized using recombinant mPlGF, and IgG was purified by two-step affinity chromatography. Identification of specific IgG against mPlGF was confirmed by ELISA assay.

Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli

Human liver carboxylesterase 1 (CES1) plays a critical role in the hydrolysis of various ester- and amide-containing molecules, including active metabolites, drugs and prodrugs. However, it has been problematic to express recombinant CES1 in bacterial expression systems due to low solubility, with the CES1 protein being mainly expressed in inclusion bodies, accompanied by insufficient purity issues. In this study, we report an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. A one-step purification with an immobilized-metal affinity column was utilized to purify His-tagged recombinant CES1. Conveniently, both denaturant and imidazole can be removed while the enzyme is refolded via buffer exchange, a dilution method. We show that the refolding of recombinant CES1 was successful in Tris–HCl at pH 7.5 containing a combination of 1% glycerol and 2mM β-mercaptoethanol, whereas a mixture of other additives (trehalose, sorbitol and sucrose) and β-mercaptoethanol failed to recover a functional protein. His-tagged recombinant CES1 retains its biological activity after refolding and can be used directly without removing the fusion tag. Altogether, our results provide an alternative method for obtaining a substantial amount of functionally active protein, which is advantageous for further investigations such as structural and functional studies.

Purification of coagulation factor VIII by immobilized metal affinity chromatography.

Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.