A truncated and dimeric format of an affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity

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© 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Biotechnol. J. 2015, 10, 1707–1718 DOI 10.1002/biot.201500131

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1 Introduction

Alzheimer’s disease (AD) is the leading cause of demen-tia worldwide, affecting approximately 35 million people [1]. Despite tremendous research efforts during the last decades, there is currently no treatment that significantly alters the course of the disease or efficiently stops its development [2–4]. According to the amyloid cascade hypothesis, the amyloid beta (Ab) peptide is central to

the pathogenesis of AD [4]. It has been hypothesized that the aggregation process of these peptides (mainly 40 and 42 amino acids in length) into oligomers, protofibrils and fibrils play a pivotal role in the neuropathology of the disorder [5, 6]. Consequently, therapeutic interventions targeting the production and aggregation, as well as clearance of Ab from the brain are under investigation [2, 7, 8]. One approach for therapy is based on admin-istration of Ab-specific agents that bind directly to Ab aggregates in the brain or free Ab peptides in the plasma (peripheral sink mechanism) [4, 9–12]. Conventional anti-bodies have demonstrated the potential of immuno-therapy, nevertheless, they have also been associated with severe side effects such as Fc-mediated proinflam-matory immune responses [13–15]. Therefore, alternative approaches using engineered antibody domains and alternative scaffold-proteins that lack effector functions have been suggested to provide safer and more effective

Research Article

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity

Hanna Lindberg1, Torleif Härd2, John Löfblom1 and Stefan Ståhl1

1 Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden

2 Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

The amyloid hypothesis suggests that accumulation of amyloid b (Ab) peptides in the brain is involved in development of Alzheimer’s disease. We previously generated a small dimeric affinity protein that inhibited Ab aggregation by sequestering the aggregation prone parts of the pep-tide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Ab peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Ab peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Ab-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Ab with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Ab1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Ab peptide.

Keywords: Affibody molecules · Affinity maturation · Amyloid beta · Bacterial display · Combinatorial protein engineering

See accompanying commentary by Erwin De Genst and Serge Muyldermans DOI 10.1002/biot.201500405

Correspondence: Prof. Stefan Ståhl, Department, Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology (KTH), AlbaNova University Center, S-106 91 Stockholm, Sweden E-mail: [email protected]

Abbreviations: ABD, albumin binding domain; A, amyloid beta; AD, Alz-heimer’s disease; FACS, fluorescence-activated cell sorting; HSA, human serum albumin; Z, affibody molecule

Received 20 MAR 2015Revised 29 MAY 2015Accepted 06 JUL 2015Accepted article online 14 JUL 2015

Supporting information available online

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therapies [8, 16, 17]. Alternative scaffold-proteins are in general much smaller than full-length antibodies and can usually be engineered into multivalent as well as mul-tispecific units with an overall size that remain smaller than the conventional antibody [17, 18]. Such features can potentially improve in vivo biodistribution of the agent [19–22]. One type alternative binding-protein that has been investigated for various diagnostic and therapeutic applications is the small three-helical bundle Affibody molecule (6.5 kDa) [17, 23].

We previously generated an Affibody molecule (denot-ed ZAb3) targeting monomeric Ab with a 17 nM affin-ity, as determined by isothermal titration calorimetry (ITC) and confirmed by surface plasmon resonance SPR [24–26]. Although this binder is based on the Affibody scaffold, it has evolved to adopt a unique and complex binding mechanism that is potentially more efficient for interactions with aggregation-prone peptides. Structural analysis has shown that two identical disulfide-linked Affibody units encapsulate one Ab peptide, and that both the Affibody domains and the Ab peptide undergo structural rearrangement upon binding. The Ab peptide folds into a b-hairpin structure, allowing the first α-helices in both Affibody molecules to adopt a b-sheet structure with unstructured N-termini [25, 27]. In a recent in vivo study using an Ab-transgenic fruit fly model of AD, it was demonstrated that the ZAb3 Affibody molecule efficiently inhibits the formation of Ab aggregates, thereby abolish-ing the neurotoxic effects and restoring the life span of the flies [25, 28].

For further potential preclinical and clinical stud-ies, we hypothesized that increasing the affinity of the dimeric Affibody protein for the Ab peptide could improve its potency. It has been shown that there is a correla-tion between the affinity of peripherally-administered Ab-specific agents and the efflux of Ab from the brain to the serum [29]. As levels of Ab peptides in the blood are low (subnanomolar) [30–34], it is probably critical to use capturing-agents of high affinity for such applications.

It has previously been demonstrated that staphylococ-cal cell surface display in combination with fluorescence-activated cell sorting (FACS) is well suited for combina-torial affinity maturation efforts, since it allows for high precision affinity discrimination between binders [35–38]. Recently, the method was used for affinity maturation of a HER3-specific Affibody, and generated binders with picomolar affinities [39].

In this study, we present the successful engineer-ing of new single-chain dimeric high-affinity variants of the complex Ab-binding Affibody molecule using the staphylococcal platform. A novel scaffold was designed, in which: (i) the two domains were formatted as a genet-ic head-to-tail dimer; (ii) the unstructured N-terminal sequence was removed; and (iii) the first part of the second domain was replaced with a hydrophilic serine-glycine linker. Importantly, head-to-tail dimeric formats of

the novel type of Ab targeting agents also allowed for cell surface display on recombinant staphylococci, followed by FACS [40]. Different randomization approaches were used for the two libraries, based on structural analysis of the interaction between the original ZAb3 Affibody and the Ab peptide [25]. DNA libraries were gener-ated using trinucleotide synthesis and subsequently dis-played on Gram-positive Staphylococcus carnosus cells. Several variants with improved affinity for Ab1-40 were isolated using flow-cytometric sorting. The best candi-date, denoted ZSYM73, demonstrated an affinity in the 300 pM-range. This corresponds to a 50-fold improvement in affinity compared to the first-generation Ab-binding Affibody molecule, which has demonstrated a therapeutic effect in a fruit fly model of AD [28], and a six-fold improve-ment as compared to a truncated head-to-tail dimeric variant of the original binder. Moreover, the new dimeric binder demonstrated retained melting temperature as well as complete refolding ability after heating. The new high-affinity Affibody was also able to capture aggrega-tion prone Ab1-42 peptides at concentrations reflecting physiologically relevant serum levels [30–32]. Altogether, we believe that these results demonstrate and motivate preclinical in vivo investigations of the new Ab-binding Affibody in mouse models with AD-Ab pathology.

2 Materials and methods

2.1 Labeling of HSA

Labeling of human serum albumin (HSA) with Alexa Fluor 647 succinimidyl ester (Invitrogen, Carlsbad, CA, USA) was performed according to the supplier’s recommenda-tions.

2.2 Library design and cloning

Two SlonoMax® head-to-tail dimer libraries of double-stranded DNA were purchased from SloningBioTechnol-ogy GmbH (Pucheim, Germany), encoding the truncated helix 1 plus helix 2 and 3 of the first Affibody domain and helix 1 and 2 of the second Affibody domain (asymmetric library: 5´-GCG GGT GGG GAG NNN NNN TAT NNN NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC TTC ATC NNN AGT TTA GAA GAT GAC CCA AGC CAA NNN GCT AAC TTG TTG GCA GAA GCT AAA AAG CTA AAT GAT GCT CAG GCG CCG GCG AGC AGCAGCAGC GGG AGC AGCAGCAGC GGG CGC GCG AGT GCG GGT CGC GAG NNN GTT TAT TTA NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC NNN ATC NNN AGT NNN GAA GAT GAC NNN AGC CAA NNN GCT AAC TT -3´; symmetric library: 5´-GCG GGT NNN GAG NNN GTT TAT NNN NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC TTC ATC NNN AGT TTA GAA GAT GAC NNN AGC CAA NNN GCT AAC TTG

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TTG GCA GAA GCT AAA AAG CTA AAT GAT GCT CAG GCG CCG GCG AGC AGCAGCAGC GGG AGC AGCAGCAGC GGG CGC GCG AGT GCG GGT NNN GAG NNN GTT TAT NNN NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC TTC ATC NNN AGT TTA GAA GAT GAC NNN AGC CAA NNN GCT AAC TT -3´, with randomized codons illustrated as NNN). The genes were flanked with XhoI and NheI restriction sites for subclon-ing into the staphylococcal vector. The libraries were PCR amplified in eight cycles using Phusion DNA polymerase (Finnzymes, Espoo, Finland), and the final PCR products were purified using a QIAquick PCR purification kit (Qiagen GmbH). Purified PCR products were digested by XhoI and NheI-HF (New England Biolabs) restriction enzymes and purified by preparative gel electrophore-sis (2% agarose gel) using QIAquick gel extraction kit (Qiagen GmbH). The S. carnosus expression vector pSCZ1 [38] was digested by the same enzymes and purified by preparative gel electrophoresis as described above. Puri-fied library fragments were ligated into the vector using T4 DNA ligase (New England Biolabs) at a 1:5 molar ratio of vector to insert, followed by phenol-chloroform extrac-tion, and ethanol precipitation for purification and con-centration of DNA fragments. Next, the library-encoding plasmids were transformed into electrocompetent E. coli SS320 (Lucigen Corporation, Middleton, USA) by elec-troporation. Individual clones were PCR amplified for subsequent library sequence validation using BigDye Thermo Cycle Sequencing reactions and an ABI Prism 3700 instrument (Applied Biosystems, Foster City, CA). Library plasmids were subsequently prepared using a Jet-star Maxi Kit (Genomed), purified by phenol-chloroform extraction and concentrated by isopropanol precipitation. Finally, the libraries (hereafter denoted Sc:ZASlib and Sc:ZSYMlib) were transformed into electrocompetent S. carnosus as previously described [41].

2.3 Cell labeling and FACS

At least ten times the library size of either library was inoculated to tryptic soy broth supplemented with yeast extract (TSB+Y; Merck, Darmstadt, Germany) as well as 20 μg/mL chloramphenicol and grown overnight at 37°C and 150 rpm. The following day, cells were harvested by centrifugation (6000 rpm, 6 min, 4°C) and washed in phos-phate-buffered saline supplemented with 0.1% Pluronic® F108 NF Surfactant (PBSP; pH 7.4; BASF Corporation, Mount Olive, NJ) before addition of C-terminally bioti-nylated Ab1-40 peptide (AnaSpec, San Jose, CA, US). Cells were incubated with gentle mixing at room temperature for 45 min until equilibrium was reached. Subsequent-ly, cells were washed with ice-cold PBSP and labelled with streptavidin conjugated with phycoerytrhrin (SAPE; Invitrogen, Carlsbad, CA, USA) as well as HSA conjugated with Alexa Fluor 647 for 30 min on ice at a concentration of 5 μg/mL and 150 nM respectively. After a final wash-

ing step in ice-cold PBSP, cells were resuspended in ice-cold PBSP prior to sorting. Cells were sorted in four cycles with an increased stringency in each cycle, using a MoFlo® Astrios (Beckman Coulter) flow cytometer. In the first cycle of selection, library cells were incubated with 50 nM C-terminally biotinylated Ab1-40 peptide, and in the second and third cycles concentrations of 20 nM and 10 nM, respectively, were used. In the last cycle, the cells were subjected to an off-rate selection procedure. In the off-rate selection, enriched library cells were incubated with 25 nM C-terminally biotinylated Ab1-40 peptide for 45 min to reach equilibrium binding, washed in ice-cold PBSP, incubated with 100 nM unlabeled Ab1-40 peptide for 6 h, and finally labeled with HSA-Alexa Flour 647 and SAPE prior to FACS sorting. In each sorting cycle, approx-imately ten times the library size was analyzed in the flow cytometer and the top fraction of cells (approximately 0.35%), with highest Ab1-40-binding to cell surface expres-sion fluorescence ratio was gated and sorted directly into TSB+Y. Sorted cells were inoculated to TSB+Y supple-mented with chloramphenicol (10 μg/mL) for overnight amplification at 37°C with 150 rpm shaking, prior to the next cycle of FACS. After the final cycle of sorting, cells were spread onto agar plates containing 10 μg/mL chloramphenicol. 96 randomly picked colonies from each sorted library were screened for subsequent sequence identification using BigDye Thermo Cycle Sequencing reactions and an ABI Prism 3700 (Applied Biosystems).

2.4 On-cell screening for A-binding

Isolated clones from the libraries were individually inocu-lated to TSB+Y with chloramphenicol (10 μg/mL) and grown overnight at 37°C and 150 rpm. 106 overnight-cultured cells were pelleted by centrifugation and washed in PBSP before resuspension in 1 nM biotinylated Ab1-40 (AnaSpec). After 45 min incubation at room temperature with gentle mixing, cells were washed with ice-cold PBSP and labeled with SAPE and HSA-Alexa Fluor 647 for 30 min on ice. Finally, cells were washed and resuspend-ed in ice-cold PBSP, prior to flow-cytometric analysis. The mean fluorescence intensities (MFI) from Ab1-40-binding and cell surface expression were measured in a Gal-lios (Beckman Coulter) flow cytometer. The head-to-tail homodimer of the first-generation binder (ZAb3)2 as well as the eleven amino acid N-terminally truncated head-to-tail homodimer of (ZAb3A12)2 were included in the analysis for comparison. The experiment was carried out in dupli-cates on different days using freshly prepared solutions.

2.5 On-cell off-rate ranking and determination

Individual staphylococcal clones were inoculated to TSB+Y with chloramphenicol (10 μg/mL) and grown overnight at 37°C and 150 rpm. 106 overnight-cultured cells were pelleted by centrifugation and washed in PBSP




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before resuspension in 25 nM C-terminally biotinylated Ab1-40 peptide (AnaSpec). After 45 min incubation at room temperature with gentle mixing, cells were washed with ice-cold PBSP, and incubated with 100 nM unlabeled Ab1-40 for 6 h. Subsequently, cells were washed with ice-cold PBSP and labeled with SAPE and HSA-Alexa Fluor 647 for 30 min on ice. Finally, cells were washed and resuspended in ice-cold PBSP, prior to flow cytom-etry. The mean fluorescence intensities (MFI) from Ab1-

40-binding and cell surface expression were measured in a Gallios (Beckman Coulter) flow cytometer. The head-to-tail homodimers (ZAb3)2 and (ZAb3A12)2 were included in the analysis for comparison. The experiment was carried out in duplicates on different days using freshly prepared solutions.

2.6 Expression and purification of soluble Affibody molecules

Eight affinity-matured Ab1-40-binding Affibody molecules and (ZAb3A12)2 were produced and purified for further characterization. The Affibody-encoding DNA sequences were amplified from colonies by PCR, using primers that introduced upstream NdeI and downstream XhoI restric-tion sites. The Affibody sequences were subsequently subcloned into the NdeI and XhoI digested expression vector pET26b+ (Novagen), generating Affibody con-structs with a C-terminal His6-tag. The plasmids were transformed into BL21 E. coli cells by heat shock. Cells were cultured in TSB at 37°C and as OD600 reached approximately 1, Affibody expression was induced by addition of IPTG to a final concentration of 1 mM. After overnight incubation at 25°C, the cells were harvested by centrifugation (4000 rpm, 8 min, 4°C). Cells were there-after lysed by sonication, and cell debris was removed by centrifugation (16 000 rpm, 20 min, 4°C). Affibody molecules were purified by IMAC using a HisPurTM Cobalt resin (Thermo Scientific, Rockford, USA) under native conditions. Thereafter, potential multimers were removed by size exclusion chromatography on an ÄKTA explorer 10 using a Superdex75 gelfiltration column (GE-Health-care) and PBS as running buffer. The molecular weight and the purity of the purified Affibody molecules were subsequently verified by LC/MS (Agilent Technologies 6520 ESI-Q-TOF) and SDS-PAGE using both reducing and non-reducing conditions. The protein concentration was determined by absorbance measurement at 280 nm.

2.7 Biosensor analysis

Binding affinities of the purified Affibody molecules to Ab were determined using a surface plasmon resonance (SPR) biosensor assay on a ProteOn XPR36 instrument (Bio-Rad Laboratories, CA, USA). N-terminally biotinylat-ed Ab1-40 peptide (AnaSpec) was injected over a Neutravi-din sensor chip (Bio Rad Laboratories) for immobilization

(immobilization levels were approximately 50 RU). Dupli-cate samples of each Affibody molecule were injected at concentrations ranging from 6.25 nM to 50 nM over immobilized Ab peptides. The flow rate was 50 μL/min and the association and dissociation was followed for 300 seconds and 2 h, respectively. HBS-EP was used as running buffer and 0.5% SDS was used for regeneration. In all experiments, subtraction of responses from each sam-ple over a blank surface was performed to minimize buffer contributions. The on- and off-rates were determined by non-linear regression to a Langmuir 1:1 model using the Proteon Manager Software (Bio-Rad Laboratories).

2.8 Circular dichroism spectroscopy

The secondary structure content of the affinity-matured Ab-binding Affibody molecule (ZSYM73) was analyzed at a concentration of 0.2 mg/mL with circular dichroism (CD) spectroscopy using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB, Mölndal, Sweden) in a cell with an optical path-length of 1 mm. A CD spectrum at 250–195 nm was obtained at 20°C. The thermal stability was measured at 221 nm while heating the Affibody molecule from 20 to 90°C (5°C/min).

2.9 Affinity chromatography using A-binding Affibody as ligand

The ZSYM73 Affibody molecule and a dimeric Affibody molecule with irrelevant specificity (denoted ZTaq and binding to Taq polymerase) were genetically fused to an albumin-binding domain (ABD) on the C-terminal by a 10 amino acid flexible linker. The ABD is a deimmu-nized variant of a previously affinity-matured 46 amino acid ABD [42]. Genes were prepared in the pET expres-sion vector, and DNA sequence verification and plasmid preparation was conducted as described above. Protein production and purification was in principle performed as previously described [42].

ZSYM73-ABD (700 μg) and (ZTaq)2-ABD were non-covalently coupled to 1 mL HSA-sepharose 4 fast flow (GE Healthcare, Uppsala, Sweden), respectively, by batch binding at room temperature for 20 min with gentle mix-ing. HSA-sepharose samples with bound Affibody mol-ecules were thereafter incubated with E. coli lysate (E. coli BL21*, protein content 1.0 g/L) spiked with 10 ng/mL Ab1–42 peptide for 1.5 h with gentle mixing. The sepharose was pelleted by centrifugation and washed with 100 mL PBS prior to elution (0.3 M HAc, pH 2.8) of the Affibody-captured Ab1-42 peptide from the HSA-sepharose. Eluates were lyophilized using SpeedVac (Savant Instruments, Milford, MA, USA). Samples and uncaptured spiked E. coli lysate were analyzed using SDS-PAGE and stained by GelCode Blue Safe Protein Stain (GE Healthcare). Molecular weights were estimated using SeaBlue2 Pre-stained marker (Invitrogen).




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2.10 Molecular modeling of the Affibody: A interaction

Molecular modeling was carried out within Swiss PDB-viewer 4.1 [43] using the solution structure of the ZAb3 Affibody in complex with Ab1-40 (PDB 2OTK) as a tem-plate. The most favorable conformations of selected side chain replacements were identified and refined by steep-est decent energy minimization using the GROMOS96 43B1 force field [44].

3 Results

3.1 Scaffold design and affinity maturation libraries

The new scaffold was designed as a single-chain head-to-tail genetic dimer to allow: (i) the disulfide-linked dimer to form more rapidly [28]; (ii) independent engineering of each Affibody domain; and (iii) recombinant display on the surface of staphylococci. Moreover, the library scaffold was designed to comprise an eleven amino acid truncation of the N-terminus, as elimination of the unstructured N-terminus of the Affibody has been shown

Figure 1. Illustration of the residues sub-jected for randomization in the two trun-cated head-to-tail linked dimeric librar-ies in complex with the Ab1-40 peptide (Protein Data Bank entry 2OTK). Affibody subunits are illustrated as blue and cyan colored ribbons. The b-hairpin forming fragment of Ab is colored in orange. (A, B) Two possible topologies for the asymmetric library in complex with the Ab peptide, as it is not known which of the N- and C-terminal subunits in the head-to-tail dimer corresponds to the E (blue) and F (cyan) subunits of ZAb3. (C) Symmetric library.




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Figure 2. (A, B) Density plots showing flow cytometry selections of the two affinity maturation libraries; (A) asymmetric library, (B) symmetric library. Fluorescence intensity on the x-axis corresponds to the surface expression level (monitored via HSA binding to a ABP expressed as an integral segment of the surface exposed proteins) and fluorescence on the y-axis corresponds to Ab-binding. The density plots are showing the staphylococcal library before flow-cytometric sorting of rounds 1, 2, 3 and 4, respectively, with regions used for gating outlined in each plot. Note that the concentration of biotinylated Ab is decreased in each sorting round, and in the last cycle an off-rate selection was performed, resulting in decreasing signal intensities on average in subsequent rounds. (C, D) Amino acid sequences of the first-generation head-to-tail Ab-binding Affibody molecule (truncated N-terminally by eleven amino acids) together with the FACS-isolated affinity matured Ab-binding Affibody molecules from (C) asymmetric library, and (D) symmetric library. The (S4G)2 linker is outlined between the two Affibody subunits. Randomized positions are indicated with a dot, and constant positions that have been mutated in the original scaffold are shown in the sorted sequences. The numbers to the right indicate how many times each clone was isolated.




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to positively affect the affinity to Ab [26, 40]. In the second domain, the original unstructured N-terminal was substi-tuted for a flexible (S4G)2 linker to allow for correct folding of the dimeric construct with minimal steric hindrance.

Based on this new scaffold, two different approaches were used for randomization of the libraries. The posi-tions that were targeted for randomization were based on structural analysis of the Affibody:Ab interaction and the sequence output from the selection of first-generation binders [24, 25]. In one library, 15 positions, asymmetrical-ly distributed on the two Affibody domains were partially subjected for randomization (asymmetric randomization). The positions targeted for randomization in the asym-metrically randomized library, hereafter denoted ZASlib, are structurally illustrated in Fig. 1A and 1B (note that two illustrations of the randomized positions in the head-to-tail dimer in complex with the Ab peptide is presented, as the topology of head-to-tail binding is not known in detail). In the other library, 16 symmetrically distributed positions (eight in each domain) were subjected for partial randomization (symmetric randomization). The positions targeted for randomization in the symmetrically rand-omized library, hereafter denoted ZSYMlib, are depicted in Fig. 1C.

A restricted diversity is usually desired when design-ing affinity maturation libraries for combinatorial engi-neering. Hence, around 50–75% of the codons for the original amino acids were combined with a mix of codons for one to four other amino acids in each randomized posi-tion, resulting in a lowered mutation frequency. To enable the construction of such complex randomized DNA oligos, the Slonomics technology was used (by former Sloning BioTechnology GmbH), in which sets of trinucleotide building blocks are combined during the production. This method provided complete freedom when selecting codons for the library and allowed randomizations with minimal biases compared to degenerate codons and error-prone PCR approaches. The designs resulted in theoreti-cal library complexities of 8.1 × 107 (ZASlib) and 2.3 × 107 (ZSYMlib) individual oligonucleotides. The library designs are outlined in Supporting information, Fig. S1.

3.2 Library construction

The oligonucleotide mixtures encoding the two libraries were subcloned in fusion to an albumin-binding protein (ABP) [41], into the staphylococcal display vector pSCZ1. Libraries were transformed into S. carnosus to gener-ate cell-displayed protein libraries with diversities of approximately 1 × 108 (ZASlib) and 1.3 × 107 (ZSYMlib). Sequence analysis of individual library members revealed a distribution of codons in accordance with the theoreti-cal design, and with no observed undesired codons (data not shown). However, about 10% of ZSYMlib contained too short (S4G)2 linkers. To verify that the libraries were functionally displayed on the staphylococcal surface,

cells were incubated with fluorescently labeled HSA and analyzed using flow cytometry. Around 60–67% of the population displayed surface-exposed recombinant pro-teins, as assessed by albumin binding (data not shown). When incubated with labeled Ab1-40, both libraries dem-onstrated a fraction of clones with retained target binding (Fig. 2A and 2B), as expected due to the low mutation frequency.

3.3 Flow cytometric sorting of affinity maturation libraries

In order to isolate high-affinity Ab-binding Affibody mol-ecules, the two staphylococcal-displayed libraries were subjected to four cycles of FACS (Fig. 2A and 2B). Staphylococcal cells were incubated with biotinylated Ab1-40 peptide for 45 min at room temperature to approach binding equilibrium. Cells were thereafter washed and incubated on ice with fluorescently labeled HSA and Streptavidin-conjugated phycoerythrin, for detection and normalization of the target binding with cell surface expression level, as described previously [38]. After addi-tional washing, cells demonstrating the highest target-binding to surface-expression ratio in the flow cytometer were gated (outlined in Fig. 2A and 2B) and isolated for amplification and subsequent rounds of sorting. In each cycle, the selection stringency was increased by chang-ing sorting parameters and gates, as well as by decreas-ing the target concentration (50 nM, 20 nM, 10 nM). In the last cycle, an off-rate selection approach was used to favor binders with the slowest dissociation. As shown by flow-cytometric analysis after each cycle of selection, the sorting resulted in enrichment of Ab-binding clones (Fig. 2A and 2B). After four cycles of sorting, individual colonies were sequenced for identification. Out of a total of 192 sequences (96 per library), 51 unique variants were identified in the output from the asymmetric library and 55 unique variants were identified in the output from the symmetric library (Fig. 2C and2D). Interestingly, one isolated clone from the asymmetric library contained a linker consisting of three S4 G repeats instead of two as in the design.

3.4 On-cell affinity screening of isolated binders

Isolated candidates from the FACS-sorted libraries were individually subjected to flow-cytometric analysis to screen for highest Ab-binding signals. In total, 20 variants from ZASlib and 17 from ZSYMlib, which were observed multiple times in the sequence analysis (including the variant with the longer linker from the asymmetric library) were included in the assay. Recombinant bacteria were incubated with 1 nM biotinylated Ab1-40 and secondary reagents as described above. The samples were sub-sequently analyzed for Ab-binding in a flow cytometer, and the ratio between Ab-binding signals and the signal




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of surface expression level (FL-2/FL-6) was determined. The original head-to-tail homodimer with N-terminal truncation (ZAb3A12)2 was included in the analysis for com-parison. All variants demonstrated higher binding-signals than the control, indicating improved binding affinity for Ab (data not shown).

3.5 On-cell off-rate ranking and determination

To limit the number of Ab-binding candidates for further analysis, all 37 unique clones were ranked by off-rate measurements whilst expressed on the staphylococcal cell surface. After incubation with biotinylated Ab1-40, unlabeled Ab peptide was added in four-fold excess and dissociation was allowed, as described above. The ratio between Ab-binding and surface expression level was thereafter determined (FL-2/FL-6). All candidates demon-strated considerably slower dissociation of the Ab peptide compared to the parental (ZAb3A12)2 Affibody (Fig. 3A). Eight variants demonstrating highest Ab-binding signals from both the affinity screening and off-rate ranking (four from each) were selected for further characterization. The selected clones are indicated by purple color in Fig. 3A.

3.6 Expression and purification of soluble Affibody molecules

Eight candidates from the on-cell ranking were sub-cloned to an expression vector and produced in E. coli as C-terminally His6-tagged dimeric binders. Proteins were purified by IMAC followed by SEC to remove potential multimeric complexes. SDS-PAGE analysis under both reducing and oxidizing conditions of the eluted fractions demonstrated pure proteins of correct size (Supporting information, Fig. S2).

3.7 Biosensor analysis off-rate and affinity

The eight purified Affibody proteins were subjected to affinity and off-rate determination using an SPR-based biosensor assay (Table 1). A dilution series of four dif-ferent concentrations of each binder was injected over a neutravidin chip surface with immobilized biotinylated Ab peptides (Supporting information, Fig. S3). The rate constants of the interactions were determined for all can-didates by non-linear regression using a one-site bind-ing model. Equilibrium dissociation constants, KD, were

Figure 3. (A) Off-rate ranking of the sorted binders on the staphylococcal cell surface, analyzed by flow-cytometry. The Affibody variants (including the original binder [ZAb3A12]2) are represented on the x-axis, and the ratio between Ab-binding (FL2 fluorescence intensity) and cell surface expression level (FL6 fluorescence intensity) is represented on the y-axis. The error bars show the standard deviation of the two independent experiments. The staples in purple indicate the eight clones selected for further characterization. (B) SPR sensorgram showing the interaction of the high-affinity ZSYM73 to biotinylated Ab1-40 on a neutravidin sensor chip. The Affibody molecule was injected in four concentrations (50, 25, 12.5 and 6.25 nM). Solid lines represent the global fit of on- and off-rates to a 1:1 Langmuir model. (C) SDS-PAGE analysis of protein fractions from ZSYM73-mediated capture of Ab1-42 peptides at a con-centration of 10 ng/mL from spiked E. coli lysate. ZSYM73-ABD (16.8 kDa) was allowed to bind to HSA-sepharose before loading the Ab1-42-spiked E. coli lysate. The gel was stained with Coomassie for visualization of protein bands. Lane 1, SeaBlue2 marker; lane 2, amyloid beta spiked E. coli lysate diluted 1:10; lane 3, eluted proteins (containing approximately 0.5 µg Ab1-42 peptide), lane 4, 0.5 µg Ab1-42 control peptide.




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calculated from best-fit on- and off-rates. The Affibody molecule ZSYM73 (Fig. 3B) demonstrated the highest affinity to Ab1-40, having an approximate dissociation constant (KD) of 340 pM. This corresponds to a 50-fold improvement in affinity relative to the published value for the first-generation binder (with a reported affinity of 17 nM) [25, 26, 28], and a six-fold improvement relative to an eleven amino acid truncated head-to-tail variant (Table 1). Dissociation was followed during 2 h, and the off-rate for ZSYM73 was determined to 3.2 × 10-5 s-1.

3.8 Circular dichroism spectroscopy

The highest affinity (ZSYM73) candidate was further investigated by analyzing the thermal stability and sec-ondary structure content using circular dichroism spec-troscopy. The analysis indicated that this Affibody mol-ecule had retained the secondary structure content of the first-generation binder [25]. To determine the thermal stability of the binder, the sample was heated from 20 to 90°C, while monitoring the helical content, resulting in melting temperature of approximately 43°C, which is similar to the first-generation variant [25]. Additionally, a CD spectrum was obtained after the variable temperature measurement in order to assess the reversibility of the unfolding after heat treatment. The spectrum revealed a perfect overlap with the spectrum from the untreated sample, strongly suggesting that the Affibody molecule completely refolded after heating to 90°C (Supporting information, Fig. S4).

3.9 Affibody-mediated A capture from spiked E. coli lysate

The ability of the new Ab-binding ZSYM73 Affibody mol-ecule to capture Ab peptides in a complex solution was analyzed in an affinity chromatography assay. In a poten-tial future therapeutic in vivo setting, the Affibody would likely benefit from fusion to an ABD since it has previously

been demonstrated that fusion of Affibody molecules to an affinity-matured variant of ABD dramatically improves the time in circulation by binding to serum albumin [19, 39, 45]. Hence, the Affibody was fused to an ABD, and the chromatography assay was designed to assess if the Affibody could capture Ab while simultaneously interact-ing with albumin. A non-relevant Affibody molecule in the same format was also included in the experiment as a negative control (data not shown). The Affibody was first bound to HSA-sepharose and thereafter incubated with the Ab-containing (approximately 10 ng/mL) bacterial lysate. This concentration should reflect physiological lev-els of Ab1-42 in the blood, even though levels are reported to vary significantly between patients [31, 32, 34]. After incubation and elution, SDS-PAGE analysis of the eluted fraction from the capture demonstrated that the ZSYM73 ligand was capable of efficient capture of Ab1-42 peptides from a complex mixture (Fig. 3C). The upper band in lane 2 corresponds the ABD-fused ZSYM73 ligand and the lower band corresponds to the Ab peptide (arrow head). Hence, both proteins were eluted from the HSA-resin at acidic conditions, as expected.

3.10 Molecular modeling

Potential conformations of the side chain replacements in ZSYM73 were modeled to understand if and how they would act to increase binding affinity to Ab. The solution structure of a first generation Affibody binder (ZAb3) in complex with Ab1-40 [25] was used as a template. ZSYM73 contains three replacements in each of the N- and C-terminal subunit sequences. These correspond to I16R, H32R and H35E in the E and F subunits of ZAb3. However, the two-fold symmetry of ZAb3 is broken in complex with Ab. The asymmetry primarily affects modeling of the I16R replacement since the side chain at position 16 is in close contact with the Ab ligand. The selection of an arginine at position 16 can be rationalized for both subunits as illustrated in Fig. 4. The I16R replacement on the E subu-

Table 1. Equilibrium dissociation constants (KD), association rate constants (ka), and dissociation rate constants (kd) for the affinity matured Ab-binding Affibody molecules, determined by SPR

Clone KD (M, mean ± SD)a) ka (M–1s–1 mean ± SD)a) kd (s–1, mean ± SD)a)

ZAb3A12 1.4 × 10–9 ± 0.1 1.6 × 105 ± 0.1 2.1 × 10–4 ± 0.02(ZAb3A12)2 2.0 × 10–9 ± 0.2 4.9 × 104 ± 0.3 9.6 × 10–5 ± 0.6ZSYM25b) 5.9 × 10–10 ± 0.4 7.2 × 104 ± 0.6 4.2 × 10–5 ± 0.05ZSYM57 1.4 × 10–9 ± 1.4 1.1 × 105 ± 0.9 1.5 × 10–4 ± 0.04ZSYM73b) 3.4 × 10–10 ± 0.0 9.4 × 104 ± 0.2 3.2 × 10–5 ± 0.04ZAS33 9.7 × 10–10 ± 0.0 9.1 × 104 ± 0.3 8.8 × 10–5 ± 0.02ZAS56 1.0 × 10–9 ± 0.1 8.8 × 104 ± 0.6 9.0 × 10–5 ± 0.04ZAS66b) 2.6 × 10–9 ± 0.9 2.8 × 104 ± 0.9 6.7 × 10–5 ± 0.09ZAS93 1.2 × 10–9 ± 0.0 1.3 × 105 ± 0.2 1.5 × 10–4 ± 0.01ZAS94b) 6.9 × 10–9 ± 6.1 1.6 × 103 ± 1.3 7.1 × 10–5 ± 0.40

a) Analyzed in duplicatesb) 7600 seconds dissociation




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nit would enable additional stabilizing hydrogen bonds with tyrosine residues (Y16) on both the E and F subunits (Fig. 4A). Similarly, I16R replacement on the F subunit would enable a hydrogen bond with Y16 on the E subunit and, probably more importantly, a salt bridge bond with a glutamate (E16) on the Ab ligand (Fig. 4B). It is likely that one or more of these hydrogen bonds favored selec-tion of I16R Affibody variants from the symmetric library. Moreover, nonpolar interactions involving the original four-carbon isoleucine side chain would in both cases to a large extent be retained by the three-carbon aliphatic part of the arginine side chains.

Replacements corresponding to I16R are prevalent also in the asymmetric library, in particular in the C-ter-minal subunit. However, this does not provide any further clues with regard to specific interactions, compared to the symmetric library, because it is not known which of the N- and C-terminal subunits in the head-to-tail dimers correspond to the E and F subunits of ZAb3.

The selection of H32R and H35E in ZSYM73 (and most of the other selected variants) can also be understood. This is because R32 and E35 can be expected to form a salt bridge that presumably acts to stabilize helix 2, which would be favorable for complex formation (Fig. 4C).

4 Discussion

4.1 Alzheimer’s disease

Development of therapies that reduce amyloidogenic levels in the brain holds promise for treatment of AD, but has not yet proven successful in the clinic [10, 16, 46, 47]. One strategy to prevent aggregation of amyloid com-plexes could potentially be by shifting free Ab levels from the brain to the periphery, according to the peripheral sink mechanism. In order to attain this shift, it would be critical to use binding agents with high affinity, as levels

of Ab peptides in the blood are low [10, 46, 48]. To meet these ends, we here set out to generate improved high-affinity variants of the previously generated Ab-specific Affibody molecule. Affibody molecules have previously been successfully investigated as diagnostic and thera-peutic agents in both preclinical and clinical studies for cancer [17].

4.2 Library design and affinity maturation

We combined a rational design strategy with a combina-torial library approach, and selected for improved variants using staphylococcal cell surface display in combination with fluorescence-activated cell sorting. First, we format-ted the original ZAb3 binder into a new single-chain head-to-tail homodimeric scaffold, comprising an eleven amino acid truncation at the N-terminus of the first domain [40]. We reasoned that this would facilitate independent engi-neering of the two moieties, allow for protein expression of heterodimeric domains on the cell surface, and make the binder less susceptible to reducing conditions. We excluded the N-terminal of the first Affibody domain in the scaffold. In the second domain, the original unstructured N-terminal was instead replaced with flexible serine-glycine linkers to allow for correct folding of the dimeric construct without any potential steric hindrance. Based on this new scaffold, two libraries were designed using different approaches for randomization. The strategies for randomization were based on previously generated NMR data on the Affibody:Ab interaction in combination with sequence analysis from the first-generation binders. In one library, an asymmetric randomization approach was employed, in which different residues in the two Affibody domains were subjected to randomization. In the other library, a symmetric randomization approach was used, in which the same residues in both Affibody domains were subjected to randomization. In order to maintain a relatively high target-binding capacity in the library, we

Figure 4. Modeling of the substituted residues in the dimeric Affibody molecule in complex with the Ab peptide. The peptide backbone of the Ab-binding b-strand and helix 2 in the Affibody subunits are illustrated as blue and cyan colored ribbons. The b-hairpin forming fragment of Ab is colored in orange. (A) Substitution of I16R on the E subunit (blue), and possible interactions of the arginine with tyrosines on both the E and F units. (B) Substitution of I16R on the F subunit (cyan), and possible interactions of the arginine with tyrosine on the F subunit and glutamate on the Ab peptide. (C) The H32R and H35E substitutions are expected to form a salt bridge that presumably acts to stabilize helix 2. The potential R32 and E35 salt bridge is shown for the F subunit for comparison with the original H32 and H35 side chains on the E subunit.




Biotechnol. J. 2015, 10, 1707–1718

aimed at a low mutation frequency with a high percent-age of the original amino acid in each randomized posi-tion. The libraries were displayed on the surface of staph-ylococci, with approximately 108 members in each library, making them the largest reported staphylococcal libraries so far. High-affinity Ab-binders were selected from the libraries using four cycles of FACS. To facilitate isolation of Ab-specific Affibody molecules with improved target-retention capacity, an off-rate procedure was employed in the last cycle of sorting. On-cell analysis of sorted bind-ers from the libraries yielded library members with sig-nificantly improved binding-capacity as compared to the first-generation binder. The equilibrium dissociation con-stants for eight candidates were determined and the best performing candidate ZSYM73 had an affinity of 336 pM. In general, the affinities of the selected binders from the two libraries were in the same order of magnitude, sug-gesting that both approaches for randomization were suc-cessful. Interestingly, one of the Affibody molecules that was generated from the asymmetric library was evolved to have a longer linker (S4G)3. We speculate that linker engineering in the ZSYM73 variant might be a future strategy for improving the affinity even further. Structural modeling of the interaction between the new agent and the Ab peptide was used to explain the positive contribu-tions from the observed mutations obtained by the com-binatorial protein engineering. Further characterization of the top-candidate ZSYM73 Affibody molecule demon-strated complete refolding after heat-treatment, which is promising for efficient labeling of binders for potential diagnostic imaging and therapy. Further increasing the affinity and/or stability of the affibody molecule in a matu-ration effort might result in an even more potent molecule. Moreover, the new binder in fusion to an ABD molecule (for increased in vivo half-life) showed efficient capture of Ab1-42 peptides from lysate while simultaneously binding to albumin, indicating that the interaction is functional in a complex mixture of biomolecules, which is promising for future evaluations in preclinical animal models.

4.3 Future outlook

An important aspect for future therapeutic use is the safety and efficacy of the molecule in vivo. Conventional monoclonal antibodies that have been tested in clinical settings for AD have been reported to induce side effects and to not efficiently pass the blood-brain barrier (BBB). Therefore, it would be interesting to investigate the new Affibody-based agent in preclinical and potentially alsoin clinical settings. Recently, an Affibody molecule targeting the human epidermal growth factor receptor 2 (HER2) proved to be both safe and efficacious in clinical trials formedical imaging of colon and gastric cancer [22]. When investigating the HER2-binding Affibody tracer in relapsing breast cancer patients, brain metastases could be readily visualized, indicating that sufficient amount

of Affibody molecules had been able to pass the BBB. Although our efforts to develop Affibody-based agents for capture of Ab-peptides is primarily focused to the peripheral compartment, clinical results from the HER2 investigation together with previous findings by other groups [49, 50] could potentially support capture of pep-tides also in the CNS.

To conclude, we believe that the dimeric format and the substantially improved affinity are valuable properties for an anti-Ab capturing agent and motivate preclinical in vivo therapeutic investigations. Moreover, we believe that the large libraries from this study could potentially be used to generate binders that sequester and hence inhibit aggregation of peptides and proteins involved in other neurodegenerative diseases.

The authors wish to thank Linnéa Pettersson and Joel Lindgren for help with protein production and purification and Dr. Bertil Macao at the University of Gothenburg for providing research reagents. The KTH Center for Applied Proteomics funded by the Erling-Persson Family Founda-tion, and the Swedish Research council (VR grant 621-2012-5336) are acknowledged for funding. Conceived and designed the experiments: H. L. T. H. J. L. S. S. Performed the experiments: H. L. T. H., J. L. Analyzed the data: H. L., T. H., J. L., S. S,. Contributed reagents/materials/analysis tools: H. L., T. H., J. L., S. S. Wrote the paper: H. L., T. H., J. L., S. S.

S. S. and J. L. are members of the Technical Advisory Board of Affibody AB, Solna, Sweden. All other authors declare no financial or commercial conflict of interest.

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EditorialBiotechnology Journal brings more than biotechnologyAlois Jungbauer and Sang Yup Lee

http://dx.doi.org/10.1002/biot.201500581

Meeting ReportPlant Science Student Conference (PSSC) 2015 – Young researchers in green biotechnologySusann Mönchgesang, Christoph Ruttkies, Hendrik Treutler and Marcus Heisters


CommentaryDevelopment of a high affinity Affibody-derived protein against amyloid β-peptide for future Alzheimer’s disease therapyErwin De Genst and Serge Muyldermans


ReviewThe long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virusMaría José Lista, Cécile Voisset, Marie-Astrid Contesse, Gaëlle Friocourt, Chrysoula Daskalogianni, Frédéric Bihel, Robin Fåhraeus and Marc Blondel


Mini-ReviewMicro 3D cell culture systems for cellular behavior studies: Culture matrices, devices, substrates, and in-situ sensing methods Jonghoon Choi, Eun Kyu Lee, Jaebum Choo, Junhan Yuh and Jong Wook Hong


ReviewOuter membrane vesicles as platform vaccine technologyLeo van der Pol, Michiel Stork and Peter van der Ley


Research ArticleA truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinityHanna Lindberg, Torleif Härd, John Löfblom and Stefan Ståhl


Research ArticleEnhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strainsAnett Patzschke, Matthias G. Steiger, Caterina Holz, Christine Lang, Diethard Mattanovich and Michael Sauer


Research ArticleA perfusion bioreactor system efficiently generates cell-loaded bone substitute materials for addressing critical size bone defects Claudia Kleinhans, Ramkumar Ramani Mohan, Gabriele Vacun, Thomas Schwarz, Barbara Haller, Yang Sun, Alexander Kahlig, Petra Kluger, Anna Finne-Wistrand, Heike Walles and Jan Hansmann


Research ArticleBiocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymesAlessandro Pellis, Enrique Herrero Acero, Hansjoerg Weber, Michael Obersriebnig, Rolf Breinbauer, Ewald Srebotnik and Georg M. Guebitz


Biotechnology Journal – list of articles published in the November 2015 issue.

Cover illustrationThis regular issue of BTJ features articles on the production of bio fuels, small molecules and recombinant proteins. The cover is inspired by an article describing increased expression levels of recombinant proteins in potato tubers upon post-harvest light treatment. © Lenslife – Fotolia.com











Research ArticleA systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon sourceAntonio Valle, Gema Cabrera, Howbeer Muhamadali, Drupad K. Trivedi, Nicholas J. W. Ratray, Royston Goodacre, Domingo Cantero and Jorge Bolivar


Research Article Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzymeTing Tang, Chongli Yuan, Hyun-Tae Hwang, Xuebing Zhao, Doraiswami Ramkrishna, Dehua Liu and Arvind Varma


Research ArticleThe potential of random forest and neural networks for biomass and recombinant protein modeling in Escherichia coli fed-batch fermentations Michael Melcher, Theresa Scharl, Bernhard Spangl, Markus Luchner, Monika Cserjan, Karl Bayer, Friedrich Leisch and Gerald Striedner


Research ArticleDetermination of antibiotic EC50 using a zero-flow microfluidic chip based growth phenotype assay Jing Dai, Sang-Jin Suh, Morgan Hamon and Jong Wook Hong


Research ArticleA potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyproteinEszter Majer, José-Antonio Navarro and José-Antonio Daròs


Research ArticlePost-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers Luis M. Larraya, Alicia Fernández-San Millán, María Ancín, Inmaculada Farran and Jon Veramendi


Biotech MethodPrediction of reversible IgG1 aggregation occurring in a size exclusion chromatography column is enabled through a model based approachFrida Ojala, Anton Sellberg, Thomas Budde Hansen, Ernst Broberg Hansen and Bernt Nilsson


Biotech MethodDual lifetime referencing enables pH-control for oxidoreductions in hydrogel-stabilized biphasic reaction systemsJens Begemann and Antje C. Spiess


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A truncated and dimeric format of an affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity