ISSN 1860-6768 · BJIOAM 2 (11) 1313–1448 (2007) · Vol. 2 · November 2007

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Biotechnol. J. 2007, 2, 1389–1398 DOI 10.1002/biot.200700053 www.biotechnology-journal.com

1 Introduction

In proteomics research major efforts are made to discovernew biomarkers for common diseases. Proteins in bodyfluids serve several important functions including trans-port, catalytic activation, humoral immunity, protease in-hibition, maintenance of oncotic pressure, and buffering.In healthy individuals, the amounts of most of these pro-teins are kept in balance. Any deviation from the normmay indicate disease, or a healing process, or may be an

effect of medical treatment. Measuring the changes in theconcentration of individual proteins or profiles thereofprovides useful diagnostic information [1]. Consequently,body fluids provide rich sources of biomarkers and are at-tractive specimens for finding diagnostic indicators for avariety of syndromes. Human plasma is potentially themost valuable sample and is attracting an increasing in-terest in proteomics research. Plasma peruses all organsin the body and the plasma proteome has been proposedto contain basically all human proteins, although some atvery low concentrations [2].

One of the major challenges in analyzing the proteomeof human plasma is the high level of complexity, with animmense dynamic range of the concentrations of proteinspresent in the sample, varying at least from pg/mL tomg/mL. Furthermore, plasma in an extremely protein-richfluid, containing over 60 mg of the total protein per milli-liter. The presence of high abundance proteins makes theanalysis of clinically relevant biomarkers complicatedsince these might be present in very low amounts. The

Research Article

Affibody-mediated transferrin depletion for proteomicsapplications

Caroline Grönwall1, 2, Anna Sjöberg3, Margareta Ramström1, 2, Ingmarie Höidén-Guthenberg3, Sophia Hober1, 2, Per Jonasson3 and Stefan Ståhl1, 2

1Department of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden2Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH),Stockholm, Sweden3Affibody AB, Bromma, Sweden

An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phagedisplay technology from a combinatorial protein library based on the staphylococcal protein A(SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transfer-rin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity wasdemonstrated as transferrin was the only protein recovered by affinity chromatography from hu-man plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-de-pletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% ofthe total transferrin content in the samples was depleted after only two cycles of transferrin re-moval, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly sug-gest a potential for the development of Affibody-based resins for the removal of abundant proteinsin proteomics analyses.

Keywords: Affibody ligand · Affinity resin · Protein engineering · Proteomics · Transferrin

Correspondence: Dr. Stefan Ståhl, Department of Molecular Biotechnolo-gy, School of Biotechnology, AlbaNova University Center, Royal Instituteof Technology (KTH), SE-106 91 Stockholm, SwedenE-mail: stefan.stahl@biotech.kth.seFax: +46-8-5537-8481

Abbreviations: ABAS-ELISA, ABD-based Affibody screening ELISA; ABD, al-bumin binding domain derived from streptococcal protein G; CSF, cere-brospinal fluid; HRP, horseradish peroxidase

Received 26 March 2007Revised 20 June 2007Accepted 22 June 2007

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most abundant protein, HSA, constitutes 55% of the totalprotein content in plasma [1] and the 22 most abundantproteins constitute more than 99% of the total protein con-tent in plasma [3]. Therefore, removal of the most abun-dant proteins would significantly increase the sensitivityin the identification of low abundant proteins that arepresent in far lower concentrations, and thus constitutean important feature in proteomics research.

Different strategies for the depletion of high abundantproteins can be considered including dye-based methodsand affinity columns [4]. Some of the desirable features ofaffinity-based methods are high specificity, to minimizethe removal of nontargeted proteins, and high stability toenable reuse and reproducibility. Antibodies are the mostcommon affinity ligands used to create capture media fordepletion purposes, and combined affinity matrices fordepletion of up to 12 of the most abundant proteins inplasma have been described [5]. Depletion of HSA in com-bination with one or several other abundant proteins inplasma has proved to considerably enhance the resolutionfor proteomics analysis using both electrophoresis andLC-MS-based techniques [5–9]. However, antibodies arefar from optimal as capturing agents for several reasons.The four-chain structure and relatively large size (approx-imately 150 kDa) make directed and efficient coupling tosolid matrices complicated and leakage of light chains byshedding from the immunoaffinity resins is a commoncomplication [10]. The large size of the affinity ligand in-creases the risk for undesired interactions. If polyclonalantibodies are used, uniform preparations cannot beguaranteed, and if mAb are to be utilized as im-munoaffinity reagents, production costs are, indeed, con-siderable. An ideal affinity ligand should be a robust, sin-gle molecule that would bind specifically to its target withhigh affinity. A small size that would simplify immobiliza-tion to the solid matrix would also be a desirable feature.

A promising alternative to antibodies in biotechnolo-gy applications is different so-called scaffold proteins[11–15], e.g., “trinectins” [16, 17], “anticalins” [18], ankyrinrepeat proteins [19], and Affibody ligands [14]. Affibodymolecules [14, 20–23] are engineered affinity ligandsbased on a 58-amino acid residue protein domain, derivedfrom the IgG-binding Staphylococcal protein A (SpA).This cysteine-free three-helix bundle domain, designatedas the Z domain [24], has been used as scaffold for theconstruction of combinatorial phagemid libraries fromwhich target-specific Affibody variants can be selectedusing for example phage display technology. Affibodymolecules have been investigated for a wide variety of dif-ferent applications such as detection reagents [25, 26], forthe inhibition of receptor interactions [27], for tumor tar-geting [28, 29], and in bioseparations [22, 30, 31]. Bindingstrengths down to picomolar affinity with impressive se-lectivity in the binding have been demonstrated [32]. Thehigh stability, low molecular weight (7 kDa), productionefficiency in Escherichia coli, and the possibility for

straightforward thiol-based directed coupling to resinsare favorable features in comparison to other affinity lig-ands [13]. Accordingly, these features make Affibody mol-ecules attractive candidates as new affinity ligands fordepletion reagents in proteomics research.

One of the most abundant proteins in plasma is trans-ferrin. Serum transferrin is together with ovotransferrinand lactotransferrin, a member of an iron-binding metal-loprotein group. Transferrin is responsible for the trans-portation of free iron in the circulation and has the abilityto bind two Fe3+ ions with high affinity. This 80 kDa gly-coprotein consists of two homologous loops with the ca-pacity of binding one iron ion each at neutral pH and re-leasing it when binding to the transferrin receptor and in-ternalizing to the acidic pH of the cell endosome [33, 34].

In this study, selection of an Affibody molecule bind-ing to the high abundant human protein transferrin, usingphage display is described. Binding properties includingspecificity and selectivity of the selected antitransferrinAffibody molecule were characterized using biosensortechnology, dot blot analysis, and affinity chromatogra-phy. Affibody-mediated depletion of HSA, IgG, and trans-ferrin was evaluated for human plasma. Furthermore, todemonstrate the general applicability of the Affibody mol-ecules, the same strategy was used for the preparation ofhuman cerebrospinal fluid (CSF). This body fluid sur-rounds the brain and is therefore frequently explored forbiomarkers of conditions related to the central nervoussystem [35]. The future applications for an antitransferrinAffibody molecule in combination with other specific Af-fibody ligands for the depletion of high abundant proteinsin human samples for proteomics applications, are dis-cussed.

2 Materials and methods

2.1 Strains, vectors, and phagemid library

The amber suppressor E. coli strain RR1!M15 [36] wasused as the bacterial host for phage production andcloning procedure. The phagemid vector pAffi1 and theconstruction of the phagemid library used in this study,Zlib2002 (3 " 109 members), has been described else-where [37]. Two expression vectors were used for the sub-cloning of selected clones: pAY442, and pAY457, bothcontaining a T7 promotor [38] and a multiple cloning site,together with a kanamycin resistance gene. In addition,the expression vector pAY442 encodes an N-terminalhexahistidyl (His6) tag and the vector pAY457 encodes aC-terminal cysteine residue. The pAY442 vector was usedfor subcloning and expression of phagemid inserts fromselected clones as monomeric Affibody proteins, whilethe pAY457 vector was used for subcloning and expres-sion of the transferrin binding Affibody molecule Z00917as a dimer. The E. coli strain BL21(DE3) (Novagen, Madi-

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son, WI, USA) was used for protein production from thepAY442 and pAY457 plasmids.

2.2 Phage selections

Preparation of phage stocks from the library (Zlib2002)and between selections was performed according to pre-viously described procedures [14, 21] using the helperphage M13K07 (New England Biolabs, Beverly, MA, USA).PEG/NaCl (PEG with NaCl) was used for phage precipita-tion, routinely yielding phage titers of about 1013 pfu permilliliter. Biotinylated holotransferrin (Sigma–Aldrich,Steinheim, Germany) was used as the target protein dur-ing selections and streptavidin coated paramagneticbeads (Dynabeads® M-280 Streptavidin; Dynal A.S., Oslo,Norway) were applied as a solid support. Four rounds ofbiopanning were performed as follows. All tubes andbeads were blocked with 5% TPBSB (PBS containing 0.1%Tween 20, 0.02% Na-azid, and 5% BSA) to avoid unspe-cific binding. To further remove unwanted binders tostreptavidin, one round of negative selection was per-formed before each round of biopanning. The phage stockwas preadsorbed on streptavidin beads for 30 min at roomtemperature and the supernatant was used as an input inthe selection round. The selection was carried out in so-lution by mixing phages and target protein in a total vol-ume of 1 mL 3% TPBSB (as above but with 3% BSA) in se-lection round 1 and 0.2 mL in round 2–4. The final con-centration of biotin-transferrin in all selection rounds was100 nM. The selection mix was incubated for 2 h at roomtemperature with continuous rotation. Thereafter, thesamples were transferred to blocked streptavidin beads, 5or 1 mg in round 1 or round 2–4, respectively, and incu-bated for 20 min to capture phage-transferrin complex.Free biotin was added to the tube for the last 5 min of theincubation (d-biotin, Sigma, 100 µL 1 mM) in order tocompete with unspecific binders. The beads werewashed with 3% TPBSB 3 min per wash with increase instringency for each round, increasing the number ofwashes (two times in round 1, five times in round 2, sev-en times in round 3, and 10 times in round four) and theamount of Tween20 in the wash buffer (0.1% Tween20 inround 1, 0.2% in round 2, 0.3% in round 3, and 0.4% inround 4). The bound phages were eluted with 500 µL50 mM glycine-HCl, pH 2.1, for 10 min at room tempera-ture, followed by immediate neutralization with 50 µL 1 MTris-HCl, pH 8.0 and 450 µL PBS. Eluted phages wereused to infect RR1!M15 cells in logarithmic growthphase. The infected cell suspensions were spread on TYEplates (tryptone yeast extract agar plates supplementedwith 2% glucose and 100 mg/L ampicillin), and incubatedovernight at 37°C. The colonies were thereafter collectedby resuspension in TSB (tryptic soy broth) culture mediasupplemented with 0.5% yeast extract and a fraction wasused for the amplification of phages, generating a newphage library. Phagemid particles were rescued from in-

fected cells using the helper phage M13K07, purified andconcentrated with PEG precipitation. The selectionprocess was monitored by titration of the phage stocksbefore selection and after elution. A serial dilution ofphage solutions was allowed to infect log phase RR1!M15cells, spread on TYE plates and incubated overnight at37°C.

2.3 ELISA for postselection screening

Affibody proteins from clones obtained after three or fourrounds of selection were produced in 96 well plates andanalyzed for their transferrin-binding activity using an al-bumin binding domain (ABD) derived from streptococcalprotein G-based in an ABD. Affibody screening ELISA(ABAS-ELISA). Randomly picked colonies (279 clonesfrom selection round 3 and 93 from round 4) were sepa-rately inoculated in 1 mL TSB medium supplementedwith 0.5% yeast extract, 100 mg/L ampicillin (Tamro, Van-taa, Finland), and 1 mM isopropyl #-D-thiogalactoside(IPTG; Apollo Scientific, Derbyshire, UK) in deep wellplates and grown with shaking overnight at 37°C. Thus,Affibody proteins from the individual clones were ex-pressed directly from the pAffi1 phagemid vector as fu-sion proteins to an ABD [39]. Cells were pelleted by cen-trifugation at 3000 " g for 10 min, the supernatant wasdiscarded and the cell pellets were resuspended in400 µL PBS containing 0.05% Tween20. The cells weredisrupted by freezing at $80°C followed by thawing in wa-ter bath. Centrifugation at 3500 " g for 20 min resulted inpelleted insoluble material and the Affibody proteins inthe supernatant. In the ELISA assay, 96 well plates(Nunc, Roskilde, Denmark) were coated with HSA (Sig-ma), 6 µg/mL in 50 mM sodium carbonate at pH 9.5 perwell overnight at 4°C. After blocking the plates with 2%low fat milk in PBS 0.05% Tween20, the crude protein su-pernatants were added and the plates were incubated1.5 h at room temperature with shaking. Transferrin bind-ing was visualized by incubation with biotin-transferrin(Sigma, 1 µg/mL for the selection round 4 clones and3 µg/mL for the clones from round 3) followed by incuba-tion with streptavidin-horseradish peroxidase (HRP)(Dako, Glostrup, Denmark, 1:5000) and detection withImmunoPure® TMP substrate (Pierce, Rockford, IL, USA).Furthermore, an ELISA for the detection of unspecificbinders to streptavidin was performed in parallel to theABAS-ELISA for the 93 clones obtained from selectionround 4. In this assay, the crude protein solution from theexpression in 96 deep well plates was added to strepta-vidin precoated microtiter plates (Nunc) blocked with0.5% casein. Affibody-binding to streptavidin was de-tected with a polyclonal rabbit antibody against all Affi-body molecules (rabbit serum L0117-73) and a goat-%-rab-bit Ig-HRP secondary antibody (Dako).

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2.4 DNA sequencing

DNA sequencing of phagemid (pAffi1) inserts was per-formed after three and four rounds of biopanning onclones that were found positive in the ELISA. Altogether,42 clones from round 4 and 18 clones from round 3 werechosen and sequenced using specific sequencingprimers and BigDye™ terminators (Amersham Bio-sciences, Uppsala, Sweden). Purified sequencing reac-tions were analyzed on ABI Prism® 3100 Genetic Analyz-er (Applied Biosystems, Foster City, CA, USA). SubclonedDNA fragments were verified using the same procedure.

2.5 DNA constructions

DNA fragments encoding five different potentially trans-ferrin-binding Affibody molecules (Z00917, Z00935,Z00936, Z00937, and Z00939) were subcloned from thepAffi1 phagemid as monomers into the expression vectorpAY442 with sticky end PCR and ligation. Correct cloneswere verified using PCR screening and DNA sequencing.The best transferrin-binding Affibody molecule, Z00917,was further subcloned as a head-to-tail dimer into the ex-pression vector pAY457, according to the same method asdescribed for the pAY442 constructs. To create dimericconstructs, the pAY457 vector with Affibody monomerwas digested with AccI and religated with the hybridiza-tion product.

2.6 Expression and purifications of His6-tag fusion proteins

Five different Affibody proteins (Z00917, Z00935, Z00936,Z00937, and Z00939) generated from the transferrinphage display selection were expressed in E. coli strainBL21(DE3) from the pAY442 expression vector, generat-ing a monomeric Affibody protein with an N-terminalHis6-tag. The His6-tagged Affibody proteins were purifiedby immobilized metal-ion affinity chromatography(IMAC) purification using the Biorobot 3000 system (Qia-gen) and Ni-NTA matrix (Qiagen) following the manufac-turer’s recommendations. The protein concentrationswere determined by amino acid analysis (Aminosyra-analyscentralen, Uppsala, Sweden) in order to get accu-rate concentrations for the biosensor analysis.

2.7 Expression and purification of (Z00917)2-cys Affibodyprotein

The antitransferrin Affibody Z00917 was expressed as adimer without affinity-tag but with a C-terminal cysteine,(Z00917)2-cys, from the pAY457 plasmid in the E. colistrain BL21(DE3). The dimeric Affibody was recoveredwith cation exchange and reversed phase chromatogra-phy using the Äkta™Explorer system (GE Healthcare,Uppsala, Sweden). An XK-16 column was prepared withSP Sepharose FF (GE Healthcare), giving a column vol-

ume (CV) of 4.5 mL and the system was equilibrated with20 mM phosphate buffer at pH 6.5. Supernatant from son-icated cells containing soluble proteins was applied to thecolumn and the unbound proteins were washed out. Thebound proteins were eluted with a gradient 0–50% of 1 MNaCl in 20 mM phosphate buffer at pH 6.5 during 20 CV.The pH of the collected eluate (in total 33 mL) was in-creased by the addition of 1.65 mL Tris-HCl at pH 8.5 andthe sample was reduced with 20 mM DTT at room tem-perature for 1 h. After the reduction, acetonitrile wasadded to a final concentration of 10% and the protein so-lution was applied to a resource RPC 1 mL column (GEHealthcare). The system was equilibrated with 10% ace-tonitrile 0.1% TFA and bound proteins were eluted with agradient 0–100% of 80% acetonitrile 0.1% TFA during40 CV. Eluted fractions were pooled and the buffer was ex-changed to PBS using a PD-10™ column (GE Healthcare).Finally, the purified protein was concentrated by ultrafil-tration (Amicon Ultra 15 mL, MWCO 10 000 Da; Millipore;Billerica, MA, USA). The identity of the purified proteinwas verified with RP-HPLC followed by MS using an Agi-lent 1100 LC/MSD system, equipped with an electrosprayionization source and single mass quadrupole detector(Agilent Technologies, Palo Alto, CA, USA).

2.8 Biosensor analyses

A Biacore® 2000 instrument (Biacore AB, Uppsala, Swe-den) was used for real-time biospecific interaction analy-sis (BIA) between selected Affibody molecules and trans-ferrin. Holotransferrin (Sigma–Aldrich) was immobilizedby amine coupling onto the carboxylated dextran layer ofa CM-5 chip (research grade), according to manufactur-er’s recommendations. Another surface was activatedand deactivated and used as a reference and human IgG(Sigma) was separately immobilized on a third flow-cellsurface of the sensor chip to serve as the negative control.Five Affibody variants originating from the transferrin se-lection (His6-Z00917, His6-Z00935, His6-Z00936, His6-Z00937, and His6-Z00939) were diluted in HBS-EP (5 mMHEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactantP20, pH 7.4) to a final concentration of 5 µM and injectedat a constant flow rate of 5 µL/min HBS-EP. In a second ex-periment the selected antitransferrin Affibody moleculeHis6-Z00917 was subjected to a kinetic analysis, in whichHis6-Z00917 protein was injected over a transferrin sur-face in different concentrations ranging from 12.5 to1000 nM with the flow rate of 30 µL/min HBS-EP. The dis-sociation equilibrium constant (KD) was estimated usingthe BIAevaluation 3.2 software (Biacore).

Furthermore, binding of the antitransferrin Affibodymolecule Z00917 to different serum proteins was studiedin order to investigate the specificity. The dimeric Affi-body (Z00917)2-cys was immobilized by thiol couplingonto the carboxylated dextran layer of a CM-5 chip (re-search grade), according to manufacturer’s recommenda-

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tions. The following human serum proteins were subse-quently injected over the surface at concentrations of10 µg/mL with the flow rate of 30 µL/min HBS-EP; holo-transferrin (Sigma), IgM (Sigma), IgG (Sigma), HSA (Sig-ma), alpha-2-macroglobulin (Sigma), IgA (Bethyl, Mont-gomery, USA), transthyretin (Merck Biosciences, Darm-stadt, Germany), hemopexin (RDI, Concord, MA, USA),and complement C4 (RDI).

For all Biacore experiments, samples were run in du-plicates and the surfaces were regenerated with 10 mMHCl after each injection. One surface on the chips in thedifferent experimental setups was activated and deacti-vated and used as a reference. The response from the ref-erence surface was subtracted from all curves when eval-uating the results.

2.9 Dot blot specificity analysis

Serum proteins were applied to 0.45 µm nitrocellulosemembrane (BioRad; Hercules, CA, USA) 1 µL per dot in aconcentration of 0.25 µg/µL. Binding to the following hu-man proteins was analyzed: HSA (Sigma), IgG (Sigma),IgM (Sigma), IgA (Bethyl), alpha-2-macroglobulin (Sig-ma), fibrinogen (Sigma), holotransferrin (Sigma), alpha-1-antitrypsin (RDI), complement C3 (RDI), haptoglobulin(RDI), alpha-1-acid glycoprotein (RDI), alpha-1-antichy-motrypsin (RDI), complement C4 (RDI), TNF-% (RDI), in-sulin (Sigma), and IgE (Nordic Biosite, Täby, Sweden).The membrane was dried and stored at room temperatureuntil analysis. The dot blot was blocked with 0.5% caseinin PBS and stained with the antitransferrin Affibody mol-ecule (Z00917)2-cys at 2 µg/mL. Furthermore, a mem-brane was prepared in parallel and incubated in PBS with-out Affibody protein as a negative control. Goat polyclon-al anti-Affibody antibodies (goat serum L0258) and sec-ondary antibody antigoat-HRP (Dako) were used fordetection. Immunoreactivity was visualized by chemilu-minescence using SuperSignal® Extended Duration Sub-strate (Pierce).

2.10 Coupling of Affibody molecules to affinitychromatography resins

The antitransferrin Affibody molecule (Z00917)2-cys, anti-HSA Affibody molecule (ABP-cys; Affibody AB, Bromma,Sweden), and anti-IgG Affibody molecule ((Zwt)2-cys; Af-fibody AB) were single point immobilized via the C-ter-minal cysteine. The Affibody proteins were reduced usingDTT for 1 h in room temperature, followed by the removalof excessive DTT using PD-10 columns (GE Healthcare).Proteins were eluted from the columns in coupling buffer(50 mM Tris-HCl 5 mM EDTA pH 8.5) and the reduced Af-fibody proteins were separately added to SulfoLink Cou-pling Gel (Pierce), 5 mg anti-HSA or anti-IgG Affibodyprotein to 1500 µL resin, and 2 mg antitransferrin Affibodyprotein to 500 µL resin. After 1 h incubation at room tem-

perature, the resins were washed with 10 mL couplingbuffer and deactivated by incubation with 50 mM freecysteine in coupling buffer for 30 min. Finally, the resinswere washed with 1 M NaCl followed by TST buffer(25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05%Tween 20, pH 8.0).

2.11 Capture of transferrin from human plasma

The antitransferrin Affibody resin was initially used indrop columns where 50 µL resin was packed in emptyPD10 columns (GE Healthcare) and used for transferrin-capture from 125 µL human plasma (L0312-50; in houseprepared pool of plasma from ten individuals) diluted 1:5in TST, thus giving a final volume of 625 µL. As a refer-ence, 1 mL TST buffer spiked with 50 µg holotransferrin(Sigma) was applied to a separate column. Columns wereequilibrated and washed with TST buffer and proteinswere eluted with 200 µL 0.5 M HAc at pH 2.8. The pH ofthe eluates was increased by the addition of 50 µL 1 MTris-base at pH 8.5.

2.12 Affibody-mediated depletion of transferrin, HSA, andIgG from human plasma and CSF samples

To evaluate the capacity of the antitransferrin resin for thedepletion of transferrin from human plasma, 60 µL humanplasma (L0312-50) diluted 1:5 in TST (giving a final vol-ume of 300 µL) was applied to 200 µL antitransferrin Affi-body resin prepared as described above. The gel slurrywas incubated for 1 h in RT with shaking. Thereafter, thedepleted plasma sample was collected, mixed with 200 µLnew antitransferrin Affibody resin, and the incubationwas repeated. The transferrin-depleted plasma was sub-sequently incubated for 1 h with a mixed anti-HSA/IgGAffibody resin containing 200 µL anti-HSA and 100 µLanti-IgG resin. Thus, giving a plasma sample depletedfrom HSA, IgG, and transferrin. For comparison, 60 µL na-tive human plasma (L0312-50) diluted 1:5 in TST buffer (fi-nal volume 300 µL) was applied to an anti-HSA/IgG Affi-body resin (200 µL anti-HSA and 100 µL anti-IgG resin).After washing the resins with TST buffer, bound proteinwas eluted from the antitransferrin resin with 2 " 200 µLelution buffer (0.2 M Glycine-HCl 0.1 M NaCl and 1 Murea, pH 2.2) and from the anti-HSA/IgG resin with2 " 300 µL elution buffer. Furthermore, the Affibody-me-diated removal of transferrin in CSF was investigated asfollows. As a first step 600 µL CSF (pool of >200 individualsamples, kindly provided by Professor J. Bergquist) waslyophilized by vacuum centrifugation and thereafter re-constituted in 100 µL TST, giving a six-fold concentrationof proteins. The concentrated CSF sample was added to167 µL anti-HSA resin and incubated end-over-end for30 min at room temperature. Flow through fraction wascollected followed by washing of the resin with 400 µLTST buffer. The two fractions were pooled resulting in

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500 µL depleted CSF. HSA-depleted CSF (250 µL) wasthen applied to a mixed anti-IgG/transferrin Affibodyresin (50 µL anti-IgG and 50 µL antitransferrin resin) andincubated end-over-end 30 min at room temperature.Handee spin columns with cellulose acetate filter (Pierce)were used for handling the affinity resin in the CSF de-pletion.

Transferrin concentrations were determined in plas-ma and CSF before and after transferrin depletion, usinghuman transferrin ELISA quantification kit (Bethyl Labo-ratories), according to the manufacturer’s instructions.

2.13 SDS-PAGE analysis

Plasma and CSF samples were analyzed with SDS-PAGEbefore and after Affibody-mediated depletion of HSA,IgG, and transferrin. Samples corresponding to 0.35 µLnative plasma or 260 µL CSF were diluted in LDS samplebuffer (Invitrogen, Carlsbad, CA, USA) supplementedwith 40 mM DTT. The samples were heated at 70°C for10 min and applied to a 4–12% NuPAGE® gel (Invitrogen).The electrophoresis was performed in MES running buffer(Invitrogen). For eluates from depletion and capture ex-periments, 10 µL protein was prepared as describedabove and applied to the gel. MultiMark® multicoloredstandard (Invitrogen) was used as the molecular weightmarker.

The SDS-PAGE gel from transferrin capture wasstained with silver staining, whereas other gels werestained with CBB according to standard methods.

2.14 Western blot analysis

Eluted protein from the antitransferrrin Affibody resin af-ter one cycle of transferrin depletion was analyzed withWestern blot. Gel electrophoresis and Western blot wereperformed according to manufacturer’s protocol (Invitro-gen). Holotransferrin (2 µg; Sigma) was applied as a posi-tive control and MultiMark multicolored standard wasused for determining the molecular weight. Protein sam-ples in LDS sample buffer (Invitrogen) supplemented with40 mM DTT were heated at 70°C for 10 min and appliedto 4–12% bis-Tris NuPAGE gels. Proteins were elec-trophoresed and transferred to 0.2 µm nitrocellulose mem-brane (Invitrogen). The membrane was blocked in 0.5%casein and stained using primary HTF-14 antitransferrinmAb (Abcam, Cambridge, UK) and secondary antibodygoat antimouse Ig-HRP (Dako). Immunoreactivity was de-tected using chemiluminescence (SuperSignal ExtendedDuration Substrate; Pierce).

2.15 In-gel tryptic digestion and protein identification using MS

In order to identify the high abundant proteins in humanplasma and CSF, bands from coomassie stained SDS-PAGE gels were excised and analyzed by MALDI-TOFMS. The excised bands were destained by subsequentlywashing in 25 mM NH4HCO3 and CH3CN twice. There-after, the gel slices were dried using a vacuum centrifugefollowed by 1 h incubation in 10 mM DTT at 57°C and 1 hincubation in 55 mM iodoacetamide at room temperature.The gel pieces were again washed subsequently with25 mM NH4HCO3 and CH3CN twice and dried. Trypsin in-gel digestion was performed by incubating the gel sliceswith 12 ng/µL porcine trypsin (Promega, Madison, WI,USA) in 25 mM NH4(HCO3) at 4°C for 30 min. The solutionwas removed and the incubation was continued in 25 mMNH4(HCO3) at 37°C overnight. Thereafter, 5 µL of a solu-tion containing 60% CH3CN, 5% HCOOH was added tothe gel slices and the peptides were extracted by sonica-tion. The samples were loaded on an MTP 384 groundsteel target (Bruker Daltonics, Bremen, Germany) usingthe dried droplet technique and %-cyano-4-hydroxycin-namic acid as the MALDI-matrix. Mass spectra wererecorded using a Biflex IV MALDI-TOF mass spectrome-ter (Bruker Daltonics) running FlexControl™. Proteinswere identified using the MASCOT software (Matrix Sci-ences, London, UK).

3 Results and discussion

3.1 Selection of a transferrin-binding Affibody molecule

A transferrin specific Affibody molecule was selectedfrom a combinatorial protein library using phage displayin vitro selection technology. The library comprising3 " 109 different Affibody variants is based on the 58-residue SpA-derived Z domain. The different members ofthe library are displayed on M13 phage particles withphage protein III as fusion partner using a 3 + 3 phagemidsystem [14, 40]. Resulting clones expressing Affibodymolecules were screened using an ABAS-ELISA for trans-ferrin binding activity after the third and fourth round ofbiopanning. The ELISA showed that 46% of the 93 ran-domly picked clones from round four (Fig. 1) and 8% of the279 clones from round three seemed to bind transferrin.Screening for binding to streptavidin gave 7.5% unspecif-ic binders in round four and no crossreaction betweenstreptavidin and transferrin binding was observed. DNAsequencing was performed on 60 ELISA-positive clones,18 from round three and 42 from round four, revealing 11unique phagemid inserts. One Affibody molecule,Z00917, occurred altogether 50 times, while the addition-al ten Affibody molecules occurred once each. All 42

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ELISA-positive clones from selection round four turnedout to be the same, namely Z00917.

Five Affibody variants from the transferrin selectionwere chosen for further characterization (Z00917, Z00935,Z00936, Z00937, and Z00939). DNA fragments encodingthe five Affibody molecules were subcloned and ex-pressed in E. coli. The purified Affibody proteins were ini-tially analyzed for transferrin binding by surface plasmonresonance biosensor technology using a Biacore instru-ment. The biosensor analysis revealed that only the Affi-body molecule Z00917 dominating in the phage selec-tions showed significant binding to transferrin, while theother Affibody molecules showed considerably weakerbinding and they were therefore not further investigated.After the initial screening, the selected Affibody moleculeZ00917 was subcloned and produced in E. coli as a head-to-tail dimer with a C-terminal cysteine. The correct massof the purified dimeric transferrin-binding Affibody mole-cule was verified by MS (data not shown).

3.2 Biosensor analysis

The binding properties of the antitransferrin Affibodymolecule (Z00917) were further investigated with biosen-sor technology using a Biacore instrument. The mono-meric Affibody molecule (His6-Z00917) was subjected tokinetic analysis by injection at different concentrationsover a holotransferrin biosensor surface. The resultingsensorgram (Fig. 2A) was analyzed with the BIAevalution3.2 software (Biacore) assuming one-to-one binding andthe dissociation equilibrium constant (KD) could be esti-mated to 400 nM by steady-state determination.

The biosensor technology was also used for studyingthe specificity of the antitransferrin Affibody molecule.The antitransferrin Affibody dimer, (Z00917)2-cys, was

single point-immobilized in a directed manner to the Bia-core biosensor surface using the C-terminal cysteine.Having a bivalent ligand immobilized on the Biacore chip,there will be no affinity contribution through avidity ef-fects. Nine different high abundant human serum pro-teins including transferrin were separately injected overthe Z00917 Affibody surface. The antitransferrin Affibodymolecule showed a strikingly high specificity to transfer-rin and gave only a low background binding to the otherserum proteins (Fig. 2B).

3.3 Specificity dot blot

A dot blot assay was performed in order to more exten-sively investigate the specificity of the antitransferrin Af-fibody molecule. Sixteen serum proteins including trans-ferrin were applied to a nitrocellulose membrane andstained with the antitransferrin dimer ((Z00917)2-cys).The Affibody molecule showed very high specificity totransferrin and in principle, no unspecific binding to the

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Figure 1. ABAS-ELISA after four rounds of phage display selection oftransferrin-binding Affibody molecules. Affibody proteins from 93 random-ly picked clones were individually produced and screened for transferrin-binding activity. The Affibody molecules fused to the 46 amino acid ABDwere captured in microtiter wells coated with HSA. Streptavidin-HRP wassubsequently used for the detection of binding to biotinylated transferrin.Absorbance for each clone measured at 450 nm is presented.

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Figure 2. Biacore binding studies. (A) Kinetic analysis of the monomericantitransferrin binding Affibody molecule Z00917. Sensorgrams were ob-tained after injection of the purified His6-Z00917 over a holotransferrinamine-coupled biosensor flow cell surface at selected concentrations:12.5 nM (filled circles), 50 nM (open squares), 100 nM (filled diamonds),200 nM (open circles), 300 nM (filled triangles), 500 nM (open triangles),800 nM (filled squares). (B) Biacore specificity analysis. Human serumproteins were separately injected over a thiol-coupled antitransferrin Affi-body (Z00917)2-cys surface at a concentration of 10 µg/mL. Sensorgramsare shown for the following proteins presented in order from higher tolower response: holotransferrin (filled squares), %-2-macroglobulin, HSA,transthyretin, complement C4, IgA, IgG, hemopexin, and, IgM. All sam-ples were run in duplicates, and the response obtained from a referencesurface has been subtracted from the curves.

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other serum proteins could be detected (Fig. 3). Somestaining of the IgM spot was noted (Fig. 3B) but the samelevel of staining was also observed on the control mem-brane (Fig. 3C) without any Affibody ligand present andwas therefore most probably due to crossreactivity of theantibodies used for detection.

3.4 Affibody-mediated capture of transferrin from plasma

The selectivity of the transferrin-binding Affibody ligandwas further demonstrated by affinity chromatography. Anantitransferrin Affibody resin was created by single pointimmobilization of the Affibody molecule His6-(Z00917)2-cys to a resin. Measurements of concentration before andafter immobilization of the Affibody ligand showed a 91%coupling efficiency. The antitransferrin resin, packed ingravity flow columns, was used for capturing transferrinfrom human plasma. SDS-PAGE analysis with silver stain-ing revealed only one band after elution from the capturein plasma or buffer spiked with transferrin (Fig. 4A) andWestern blot confirmed the identity (Fig. 4B). Hence, theantitransferrin Affibody ligand proved to have a very highlevel of selectivity and specificity in a complex samplesuch as human plasma and should thus be of interest tofurther evaluate as a ligand for transferrin depletion of bi-ological samples.

3.5 Depletion of HSA, IgG, and transferrin from plasma and CSF

The main future application for the transferrin-bindingAffibody molecule is most likely for the depletion of trans-ferrin from human samples for proteomics research.Therefore, the use of the Affibody molecule for depletionwas evaluated by the depletion of transferrin in combina-tion with Affibody-mediated depletion of HSA and IgG inhuman plasma samples. First, transferrin was capturedfrom plasma by incubation with the antitransferrin Affi-body resin (60 µL plasma to 2 " 200 µL resin). This was fol-lowed by incubation with a combined anti-HSA and anti-IgG Affibody resin to remove HSA and IgG. The concen-tration of transferrin in the samples before and after de-pletion was determined by a transferrin-specific ELISAquantification kit. It was demonstrated that 85% of the to-tal transferrin content in the samples was depleted afteronly two cycles of transferrin removal (data not shown).Furthermore, the degree of depletion after one cycle of de-pletion was determined to be 65% giving a theoretical de-pletion efficiency after two rounds of removal of approxi-mately 88%, which is consistent with the observed ex-perimental value. SDS-PAGE analysis was used to assessthe efficiency of depletion from plasma (Fig. 5A) and CSF(Fig. 5B). In the plasma depletion experiment, the deple-tion of IgG and HSA (Fig. 5A, lane 2) was evidently effi-cient, as these proteins were proven not to correspond tothe more abundant remaining bands. Upon transferrin de-pletion, a vast majority of the transferrin was captured(Fig. 5A, lane 3), and only a faint band could be observedat the size corresponding to transferrin (arrow). Encour-aged by these results, a CSF depletion experiment wasalso performed (Fig. 5B). HSA was first depleted from CSF(Fig. 5B, lane 2), however, not as efficiently as in the plas-

Figure 3. Specificity dot blot assay for the antitransferrin Affibody mole-cule Z00917. (A) Sixteen human proteins were applied to nitrocellulosemembranes according to the schematic figure in panel A in the followingorder: 1, HSA; 2, IgG; 3, IgM; 4, IgA; 5, %-2-macroglobulin; 6, fibrinogen;7, holotransferrin; 8, %-1-antitrypsin; 9, complement C3; 10, haptoglobu-lin; 11, %-1-acid glycoprotein; 12, %-1-antichymotrypsin; 13, complementC4; 14, TNF-%; 15. insulin; 16, IgE. (B) The membrane was incubated withthe dimeric Affibody ligand (Z00917)2-cys and subsequently allowed to re-act with a goat-antiAffibody serum and a secondary HRP-labeled antigoatantibody. Transferrin-binding can be observed in spot number 7. (C) Con-trol membrane treated identically but with the Affibody ligand omitted.

Figure 4. Capture of transferrin using an antitransferrin Affibody affinityresin with the transferrin binding Affibody molecule (Z00917)2-cys. (A)SDS-PAGE analysis eluted protein fractions from Affibody-mediated cap-ture of transferrin. The gel was stained by silver staining. Lane 1, eluatefrom the control sample, transferrin-spiked TST. Lane 2, eluate from hu-man plasma. (B) Western blot analysis of eluted protein after transferrindepletion of human plasma. Figure shows membrane stained with the an-titransferrin mAb HTF-14. Lane 1, reference sample, 2 µg holotransferrin.Lane 2, eluate from one round of transferrin depletion of human plasma.

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ma experiment. This is likely to be due to nonoptimal con-ditions that probably could be optimized. Subsequently,efficient capture of IgG and transferrin was achieved (Fig.5B, lane 3). ELISA analysis demonstrated that the deple-tion efficiency of transferrin from CSF was approximately78% using only one round of Affibody-mediated removalof transferrin (data not shown).

4 Concluding remarks

An Affibody ligand with specific binding to human trans-ferrin was selected by phage display technology. In fact,among the selected Affibody variants the dominatingclone was the only one showing strong transferrin bind-ing (KD estimated to be 400 nM for the monomer). Selec-tive binding of the acquired Affibody ligand to transferrinwas demonstrated using biosensor technology, dot blotanalysis, and affinity chromatography. Impressive speci-ficity was demonstrated and no crossreactivity was ob-served. Upon affinity capture from human plasma, trans-ferrin was the only recovered protein visualized on a silverstained gel. To demonstrate the suitable use as a ligandfor depletion in proteomics applications, Affibody-medi-ated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and CSF.For plasma, 85% of the total transferrin content in thesamples was depleted after two cycles of transferrin re-

moval, and for CSF 78% efficiency was obtained in a sin-gle-step depletion. These results clearly suggest a poten-tial for the development of Affibody-based resins for theremoval of abundant proteins for proteomics analyses.

Professor Jonas Bergquist, Uppsala University, is ac-knowledged for providing the CSF sample.

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Figure 5. SDS-PAGE analysis after Affibody-mediated depletion of HSA,IgG, and transferrin from human samples using affinity resins thiol-cou-pled with anti-HSA Affibody molecule (ABP-cys), anti-IgG Affibody mole-cule ((Zwt)2-cys), and antitransferrin Affibody molecule ((Z00917)2-cys).The identities of high abundant protein bands were determined by in-geldigestion and MALDI-TOF MS. Arrowheads indicate transferrin. (A) De-pletion of human plasma. Lane 1, undepleted plasma. Lane 2, HSA andIgG depleted plasma. Lane 3, HSA, IgG, and transferrin depleted plasma.Protein bands were identified as: I, %-2-macroglobulin; II, transferrin;III, HSA; IV, IgG; V, haptoglobulin; VI, IgG; VII, complement C3; VIII,%-1-antitrypsin; IX, fibrinogen; X, apolipoprotein A1. (B) Depletion of hu-man CSF. Lane 1, undepleted CSF. Lane 2, HSA depleted CSF. Lane 3,HSA, IgG, and transferrin depleted CSF. Protein bands were identified as:I, transferrin; II, HSA; III, IgG; IV, IgG; V, transthyretin; VI, cystatin C;VII, #-2-microglobulin; VIII, prostaglandin-H2 D-isomerase; IX,apolipoprotein A1.

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