Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.     

Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.     

Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography

Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+)-iminodiacetic acid (IDA) agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+)-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.     

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.     

Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography

In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.     

Use of Streamline chelating for capture and purification of poly-His-tagged recombinant proteins

Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6×His-tagged protein binds to a chelating resin charged with metal ions such as Ni²⁺, Cu²⁺ or Zn²⁺, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6×His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.     

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

N‐terminal processing of affinity‐tagged recombinant proteins purified by IMAC procedures

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S‐transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine‐containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7‐triazacyclononane (tacn). The use of this tag‐tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli‐expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP‐1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI‐TOF MS analysis of the cleaved products from the DAP‐1 digestion of the recombinant N‐terminally tagged proteins confirmed the complete removal of the tag within 4‐12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn‐based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli‐expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.     

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.     

Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor

The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an alpha-helical content of the purified recombinant protein of approximately 54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties.     

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.     

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.     

HaloTag-based purification of functional human kinases from mammalian cells

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.     

A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.     

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N‐terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site‐directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1. These tags were specifically developed for application with borderline metal ions, such as Ni²⁺ or Cu²⁺ ions, chelated to the immobilized ligands, 1,4,7‐triazacyclononane (tacn) and its analogs. Due to the ability to control cleavage site structure and accessibility via site directed mutagenesis methods, these procedures offer considerable scope to obtain recombinant proteins with authentic native N‐termini, thus avoiding any impact on structural stability, humoral and cellular immune responses, or other biological functions. Collectively, these IMAC‐based methods provide a practical alternative to other procedures for the purification of recombinant proteins with tag removal. Overall, this approach is essentially operating as an integrated down‐stream purification capability.     

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.     

Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied - studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)(2)-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members.