Ribosome Display: In Vitro Selection of Protei.n-Protein Interactions
Ribosome display is an in vitro technology to identify and evolve proteins or peptides binding to a given target (Fig. 1) (Hanes et al., 2000a). While most selection technologies need living cells to achieve the essential coupling of genotype and phenotype, ribosome display uses the ribosomal complexes formed during in vitro translation to generate the physical coupling between polypeptide (phenotype) and mRNA (genotype) (Amstutz et al., 2001). Hence, no transformation step limiting the size of the usable library is necessary, allowing the selection from very large combinatorial libraries. In addition, the rapid selection cycles require an integral polymerase chain reaction (PCR) step, which can be used for randomization, making this method ideal for directed evolution experiments. The fact that the ribosomal complex used for selection is not covalent allows an uncomplicated separation of the mRNA from the selected ribosomal complexes, even if the selected molecules bind the target with very high affinity or are even trapped covalently (Amstutz et al., 2002; Jermutus et al., 2001). All these benefits make ribosome display a good alternative to other selection techniques, such as phage display (Smith, 1985).
Ribosome display has been applied successfully for the selection of peptides (Matsuura and Plückthun, 2003; Mattheakis et al., 1994), as well as folded proteins such as antibody fragments (Hanes and Plfickthun, 1997; He and Taussig, 1997; Irving et al., 2001). Ribosome display can also be considered for the screening of cDNA libraries for interaction partners. Ribosome display ultimately selects always for a specific binding event. However, by designing the selection pressure carefully, molecules can be selected for many other parameters, such as enzymatic turnover (by selection with a suicide inhibitor, or active site ligand) (Amstutz et al., 2002; Takahashi et al., 2002), protein stability (by selecting for binding under conditions where most library members will not fold) (Jermutus et al., 2001), or protein biophysical properties (resistance to proteases and nonbinding to hydrophobic surfaces) (Matsuura and Plfickthun, 2003). It is the combination of this array of selection pressures with the convenient PCR-based randomisation techniques that makes ribosome display a powerful and versatile technology.
II. MATERIALS AND INSTRUMENTATION
The following chemicals and enzymes are necessary to prepare the extract and to perform ribosomedisplay selections: Luria broth base (GibcoBRL 12795- 084); agarose (Invitrogen 30391-023); glucose (Fluka 49150); potassium dihydrogen phosphate (KH2PO4, Fluka 60230); dipotassium hydrogen phosphate (K2HPO4·3H2O, Merck 1.05099.1000); yeast extract (GibcoBRL 30393-037); thiamine (Sigma T-4625); Tris (Serva 37190); magnesium acetate (MgAc, Sigma M-0631); potassium acetate (KAc, Fluka 60034); L-glutamic acid monopotassium salt monohydrate (KGlu, Fluka 49601); 20 natural amino acids (Sigma LAA-21 kit); adenosinetriphosphate (ATP, Roche Diagnostics 519 987); phosphoenolpyruvate trisodium salt (PEP, Fluka 79435); pyruvate kinase (Fluka 83328); GTP (Sigma G-8877); cAMP (Sigma A-6885); acetylphosphate (Sigma A-0262); Escherichia coli tRNA (Sigma R-4251); folinic acid (Sigma 47612); PEG 8000 (Fluka 81268); 1,4-dithiothreitol (DTT, Promega V3155); sodium chloride (NaCl, Fluka 71376); Tween- 20 (Sigma P-7949); neutravidin (Pierce 31000); bovine serum albumin (BSA, Fluka 05476), Sacchoromyces cerevisiae RNA (Fluka 83847); ribonuclease inhibitor RNasin (Promega N211B); reverse transcriptase Stratascript (50U/µl, Stratagene 600085-51); 10× Stratascript buffer (Stratagene 600085-52); dNTPs (5 mM of each dNTP, Eurogentec NU-0010-50); DNA polymerase for PCR (e.g., Vent polymerase, NEB M0254L); PCR buffer (e.g., thermopol buffer, delivered with Vent polymerase), dimethyl sulfoxide (DMSO, Fluka 41640); NTPs (50mM, Sigma); nitrocefin (Calbiochem 484400); HEPES (Sigma H-3375); spermidine (Sigma S-2501); T7 RNA polymerase (NEB M0251L); lithium chloride (LiCl, Fluka 62476); 100% ethanol (EtOH); sodium acetate (NaAc, Fluka 71180); heparin (Fluka 51550); disodium ethylenediaminetetraacetate (EDTA, Fluka 03680); T4 DNA ligase (MBI Fermentas EL0011); 4-morpholinopropanesulfonic acid (MOPS, Fluka 69949); boric acid (Fluka 15660); guanidine thiocyanate (Fluka 50990); N,N-dimethyl formamide (Sigma Aldrich 27.054-7); 37% formaldehyde (Fluka 47629); UHP water; if proteins are displayed that depend on the correct formation of disulfide bonds, protein disulfide isomerase should be used (PDI; Sigma P3818); [35S]methionine (PerkinElmer NEG009H); triethylamine (Sigma-Aldrich 90335); OptiPhase2 scintillation liquid (PerkinElmer 1200-436).
B. Bacterial Strain and Plasmid
We use E. coli strain MRE600 for the preparation of the extract. This strain is RNase I deficient (Kushner, 2002) and does not contain any antibiotic resistance (Wade and Robinson, 1966).
Ribosome-display vector (pRDV), containing β- lactamase as insert (gene bank accession: AY327136).
C. Laboratory Equipment and Hardware
The following material is used in ribosome display: ART filter pipette tips (10 µl, 20 µl, 200 µl, 1000 µl, nucleic acid and nuclease-free tips, Molecular Bioproducts); QIAquick PCR purification and gel extraction kit (QIAgen 28104 and 28704); Maxisorp plate (Nunc-Immuno plate, Nunc 430341); step pipette (Eppendorf Multipipette Plus 4981 000.019) with 5- and 10-ml tips (Eppendorf 0030 069.250 and 0030 069.269); plastic seal (Corning Inc., Costar® 6524); RNase-free 1.5-ml reaction vials (MolecularBioProducts 3445); Roche high pure RNA isolation kit (Roche 1 828 655); 0.2-mm syringe filter (Millipore SLGPR25KS); dialysis tubing with a molecular weight cutoff of 6000-8000Da (e.g., Spectrum Laboratories SpectraPor 132 650).
Furthermore, standard laboratory equipment is needed, such as Sorvall RC-5C Plus centrifuge with rotors SS-34 and GS-3 or equivalent; refrigerated table centrifuge; shaker incubator; 5-liter and 100-ml baffled shake flasks for E. coli culture; Emulsiflex (Avestin, Canada) or French Press (American Instrument Company, AMINCO); 4°C room; liquid nitrogen (N2), ELISA plate shaker; UV/VIS spectrophotometer; agarose gel electrophoresis system; latex gloves, Speed-Vac (Savant Speed Vac Concentrator SVC100H); -20 and -80°C freezer; Scintillation counter.
Preparation of S30 Extract
General considerations: 1 liter E. coli culture yields approximately 8 ml extract, if you plan to do ribosome display at a large scale, grow several cultures in parallel. It is important that the cells used for extract preparation are harvested in an early logarithmic phase. If the libraries used for selection contain disulfide bonds, one should omit DTT from the extract. If no disulfides need to be formed, 1mM DTT can be added to the S30 buffer as it increases the translation efficiency slightly.
Preparation of the S30 extract is performed according to Lesley, Zubay, and Pratt, with minor modifications (Chen and Zubay, 1983; Lesley, 1995; Pratt, 1984; Zubay, 1973).
The following buffers are used in standard ribosome- display selection rounds and we advise preparing stocks: Tris-buffered saline (TBS; 50mM Tris-HCl, pH 7.4, at 4°C; 150mM NaCl), TBS with Tween [TBST; TBS with 0.05% (500µl/l) Tween-20], washing buffer with Tween (WBT; 50mM Tris-acetate, pH 7.5, at 4°C; 150mM NaCl; 50mM MgAc; 0.05% Tween-20) and elution buffer (EB; 50mM Tris-acetate, pH 7.5, at 4°C; 150mM NaCl; 25 mM EDTA); 10× MOPS (0.2M MOPS, pH 7, 50mM sodium acetate, 10mM EDTA); 10× TBE buffer (89mM Tris-buffered saline, 89mM boric acid, 10 mM EDTA).
Note: The washing buffer used for ribosome display can be adjusted to any particular requirements given by the target molecule. Different buffer salts and detergents are compatible, only the Mg2+ concentration should be held at around 50 mM. If a new buffer composition is applied, a ribosome-display test selection round with a known binder is recommended to determine compatibility.
αtssrA: (200 µM, 5'-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA-3', standard quality)
T7B: Forward RD primer. Introduces T7 promotor and part of the 5' loop
SDplus: Forward RD primer. Introduces the Shine- Dalgarno sequence and connects the T7 promoter with the FLAG tag: (100µM, 5'-AGACCACAACGGTTTCCCAATAATTTTGTTTAACTTTAAGAAGGAGATATAT
tolAk: Reverse primer for RD used with tolA as spacer introducing a stabilizing 3' loop
RDlinktolA: (100µM, 5'-GGGGAAAGCTTTATATGGCCTCGGGGGCCGAATTCGAATCTGGTGGCCA
Primers (reverse and forward) specific for the library of interest, which must introduce appropriate restriction sites for ligation into the ribosome-display vector.
Solutions, Strain, and Hardware
Escherichia coli strain MRE600 (Wade and Robinson, 1966); Luria broth base; incomplete rich medium: 5.6 g/liter KH2PO4, 37.8 g/liter K2HPO4·3H2O,10 g/liter yeast extract, 15 mg/liter thiaminemafter autoclaving, add 50ml 40% (w/v) glucose sterile filtered; 0.1M MgAc; 10× S30 buffer: 100mM Tris-acetate, pH 7.5, at 4°C, 140mM MgAc, 600mM KAcmstore at 4°C or chill buffer in ice bath before use; 10ml preincubation mixmmust be prepared immediately before use: 3.75ml 2M Tris-acetate, pH 7.5 (at 4°C), 71µl 3M MgAc, 75µl amino acid mix (10mM of each of the 20 natural amino acids), 0.3 ml 0.2M ATP, 0.2 g PEP, 50 U pyruvate kinase.
Material: 5-liter baffled flasks; shaker at 37°C for E. coli culture; refrigerated centrifuges (GS-3, SS-34); dialysis tubing MW cutoff 6000-8000Da; emulsiflex or French press.
Do wear gloves during all following steps!
B. Premix Preparation and Extract Optimization
The premix provides the S30 extract with all amino acids (except for methionine, see later), tRNAs, the energy regeneration system, and salts, which are needed for translation. For optimal translation efficiency, every premix should be adjusted to fit the corresponding extract, especially with respect to the concentration of magnesium, potassium, PEG-8000, and amount of extract. The premix A recipe given later contains only minimal concentrations of KGlu, MgAc, and PEG-8000. By performing translations (compare Section III,F) using a test mRNA and by gradually adding increasing amounts of these components, the translation efficiency of the extract will be optimized to its maximal activity. The optimization of the premix to the $30 extract is optimally done by translating the mRNA encoding an enzyme, whose activity is determined easily, such as β-lactamase. We routinely use a cysteine-free version of the enzyme (Laminet and Plfickthun, 1989) in a ribosome-display suitable format (described in Section III,C) for optimization.
You will need approximately equivalent amounts of premix and extract. Premix A: 250 mM Tris-acetate, pH 7.5, at 4°C, 1.75 mM of each amino acid except methionine, 10 mM ATP, 2.5 mM GTP, 5 mM cAMP, 150 mM acetylphosphate, 2.5mg/ml E. coli tRNA, 0.1mg/ml folinic acid, and 4 µM α-ssrA DNA. KGlu (180-220 mM), MgAc (5-15mM), and PEG-8000 [0-15% (w/v)] have to be adjusted to the corresponding extract. β- Lactamase assay buffer: Dissolve 5.3 mg nitrocefin in 250µl DMSO and add this to 50ml 50mM potassium phosphate buffer (pH 7) (Laminet and Pliickthun, 1989).
To avoid RNase contamination, use filter tips and wear gloves for all of the following steps.
C. Preparation of the Ribosome-Display Construct
To perform ribosome display, one needs a highquality library in the appropriate format. This section does not explain how to generate this library, as this depends entirely on the experimental goal, but rather how to convert an existing one into a format suitable for ribosome display.
A ribosome-display construct is composed of a T7 promoter, followed by a ribosomal-binding site and an open reading frame, which in turn consists of the library fused in frame to a C-terminal spacer polypeptide that has no stop codon (Figs. 2 and 3). The lack of a stop codon prevents the binding of the termination factors TF-1, TF-2, and TF-3. A high magnesium concentration "sinters" the ribosome, which consists largely of folded RNA with a protein coat. The low temperature presumably prevents the hydrolysis of the peptidyl-tRNA and minimizes mRNA degradation. All these measures together ensure that the ternary complex of mRNA (genotype), ribosome, and displayed protein (phenotype) remains stable. The Cterminal spacer (usually derived from tonB, tolA, M13 gpIII or pD), which will partially remain in the ribosomal tunnel, ensures that the library protein can fold and is displayed on the ribosome. The T7 promoter allows efficient in vitro transcription of the construct. A 5' and a 3' stem loop protect the mRNA against exonucleases.
Solutions, Plasmids and Strains
QIAquick PCR purification and gel extraction kit; pRDV; appropriate restriction enzymes; T4 DNA ligase.
1. Generation of the Ribosome-Display Construct via pRDV
To accelerate the procedure of bringing a library into the ribosome-display format, we generated a vector containing the necessary flanking regions (ribosome-display vector, pRDV; Fig. 2). The library is PCR amplified, cut with the appropriate restriction enzymes, and ligated into the vector such that it is in frame with the spacer (Fig. 3). A second PCR on this ligation product directly amplifies the library with all features necessary for ribosome display: the T7 promoter, the RBS, and the spacer without stop codon (Fig. 2). This PCR product is used directly for in vitro transcription to yield the library mRNA ready to go. The main advantages of the ribosome-display vector are that it can be generated in large amounts (mini to maxi prep), it is easy to handle, and always provides error-free library flanking regions. The use of the vector is not only interesting for the initial generation of the ribosome-display construct, but also for the first selection rounds. If one only amplifies the library gene of the selected clones after the panning procedure without all the flanking regions, one is able to even recover library members partly degraded by RNases in the flanking regions. The recovered genes are then religated into pRDV and one is again ready to go for another round of selection.
2. Generation of the Ribosome-Display Construct via Assembly PCR
In some cases, it may be preferable not to ligate the library into pRDV, but to use PCR assembly to generate the ribosome display construct (Fig. 2). In this case, both the library and the spacer (e.g., tolA) are PCR amplified so that the 3' end of the library and the 5' end of the spacer share overlapping sequences (Fig. 2). The PCR products of the library and the spacer are assembled and amplified with the primers SDplus (introducing the ribosome-binding site and the connection to the T7 promoter) and tolAk. A final PCR reaction with the primers T7B (introducing the T7 promoter) and tolAk completes the construct, ready for transcription (Fig. 2).
D. Transcription of PCR Products
Solutions and Hardware
5× T7 polymerase buffer: 1M HEPES-KOH, pH 7.6, 150 mM MgAc, 10 mM spermidine, 200 mM DTT, NTPs (50mM each); T7 RNA polymerase; RNasin; 6M LiCl; 70% EtOH; 100% EtOH; 3M NaAc; agarose; guanidine thiocyanate; formamide; 37% formaldehyde; MOPS; Speed-Vac; heating blocks; UV/VIS spectrophotometer.
To avoid RNase contamination, use filter tips and wear gloves for all of the following steps.
E. Target Molecule Immobilization
To perform a selection, the target molecule must be immobilized in a conformation relevant for further applications. Typically, a protein will have to be in its native conformation. A very promising way to achieve this to biotinylate the target molecule and immobilize it via neutravidin or streptavidin. Biotinylation can be done chemically with commercially available reagents, either attaching the biotin to cysteine or lysine residues. The problem of this unspecific approach is that biotinylation might destroy epitopes. Alternatively, the target protein can be expressed in recombinant form with a biotinylation tag, i.e., a peptide sequence, which is recognized and biotinylated by the E. coli biotinylation enzyme BirA (Schatz, 1993). If for any reason biotinylation is not an option, one can either immobilize the target molecule directly on the hydrophobic surface of a microtiter plate well or use a specific antibody, which itself can be immobilized easily via protein A or G. Note that the buffers used here may have to be adapted to the needs of the target molecules.
Solutions and Hardware
TBS, TBST, WBT at 4°C
Maxisorp plate; plastic seal; step pipette; plate shaker; 20µM stock neutravidin; BSA; target molecule of choice
To avoid RNase contamination, use filter tips and wear gloves for all of the following steps. We also recommend carrying out the selection, RT and PCR in duplicate, to check the reproducibility of the selection. Therefore, one target molecule is routinely immobilized in two wells.
Day 2 (Day of the Ribosome-Display Round)
F. In Vitro Translation
UHP water; WBT; WBT with 0.5% BSA; heparin (200 mg/ml); methionine (200mM); S30 extract; premix Z; the mRNA of the library; optionally PDI (4 mg/ml, reconstituted from lyophilized protein); all buffers should be kept on ice, the RNA in liquid nitrogen unless stated otherwise
We recommend carrying out the panning in duplicate to check the reproducibility of the selection. To avoid RNase contamination, use filter tips and wear gloves for all of the following steps.
Solutions and Hardware
WBT; EB; yeast RNA (25µg/µl); cold room; microtiter plate shaker; Roche High Pure RNA isolation kit
All the following steps should be performed in a cold room (at 4°C or slightly below). The low temperature guarantees complex and mRNA stability. Under such conditions ribosomal complexes have survived off-rate selection procedures of 10 days and longer. During all binding and elution steps, shake the microtiter plate gently. To avoid RNase contamination, use filter tips and wear gloves.
Solutions and Hardware
Roche High Pure RNA isolation kit; 100mM DTT; RNasin; Stratascript (50U/µl; Stratagene); dNTPs; DNA polymerase; oligonucleotides; 10× Stratascript buffer; polymerase buffer; DMSO; agarose
The RNA purification is done according to the Roche protocol, with slight modifications. All the centrifugation steps are carried out at 4°C.
RT-PCR. If the RT-PCR with the forward (e.g., T7B) and reverse (e.g., tolAk) primers does not yield a high-quality product, one can amplify only the coding region of the selected library members. This usually improves the yield and the quality of the PCR product. This is most likely due to the fact that RNases degrade the mRNA from the ends. Amplifying only the central (library) stretch can rescue partly degraded clones. The PCR product of the library stretch is subsequently religated into pRDV as described in Section III,C. As this procedure rescues more clones than the PCR of the whole construct, we would recommend it for the first rounds to guarantee that no binders are lost.
Panning Controls. Ribosome display has many error-sensitive steps. It is therefore recommended to do panning, RT, and PCR in duplicate to check the reproducibility of the selection. Specific binders should be enriched from round to round. This should correlate with the number of PCR cycles needed to amplify the DNA after RT, which should decrease from round to round. Usually, one can reduce the cycle number by around five per round. If the selection pressure is increased, the yield will drop. When enrichment is observed (for designed ankyrin repeat protein libraries, (Binz et al., 2004) e.g., after round two, while for antibody libraries after round three to four), one can test the specificity of the selected pool by comparing panning results against the correct and an unrelated target molecule. If the pool is specific, only the correct target molecule will give a PCR product after RT. If the pool also gives a signal with the unspecific target molecule, the majority of the clones are still unspecific. However, there may also be a population of specific binders in the pool. An additional panning round with increased prepanning can reduce the background. Alternatively, single clone analysis might directly yield specific binders.
I. Radioimmunoassay (RIA)
Radioimmunoassay is another fast and convenient method to check whether specific target-binding molecules have been enriched in a pool (Hanes et al., 1998). It can be used for the evaluation of both selected pools and individual binders. RNA of a pool or single binder is translated in the presence of radioactively labelled [35S]-methionine. Therefore, the radioactive protein that binds to the surface-immobilized target molecule can be quantified easily. The binding should be performed in the presence and absence of soluble competitor target molecules, and a control for unspecific binding should be included. In the competition assay, the minimal concentration of competitor still leading to half-inhibition of the maximal binding signal is a crude measure for the affinity. Therefore, RIA facilitates the ranking of the affinities of different clones isolated after affinity maturation.
Solutions and Hardware
The general handling is the same as for the in vitro translation. However, the radioactive material must be handled with the appropriate precautions. Do the radioactive work in a designated area of your laboratory. We recommend using filter tips and wearing gloves during the experiment. We recommend doing the RIA in duplicate.
UHP water; WBT; WBT with 0.5% BSA; milk powder; heparin (200mg/ml); [35S]-methionine (10mCi/ml, 1175Ci/mmol); S30 extract; premix Z; mRNA of a selected pool or single binders; pRDV; optionally PDI (4mg/ml); 0.1M triethylamine; liquid scintillation cocktail "OptiPhase2"
Scintillation counter, ELISA plate shaker
This section states some observations we have made during the years of performing ribosome display and gives a summary of how ribosome display can be used for directed evolution experiments.
A. Selection from Naive Libraries
Naïve libraries, in our hands synthetic antibody libraries or designed repeat protein libraries, are a difficult challenge for selection experiments. The task is to select the specific binders, which are few in numbers, out of a very large number of nonbinders. The outcome of such experiments is not only dependent on the presence of high-affinity binders in the library, but also on the behavior, or "stickiness," of the rest of the library population. In other words, selection must be directed toward specific binding, in contrast to nonspecific binding. This can be achieved with experimental tricks, such as introducing a prepanning step, or by trying to reduce the stickiness of the library population. For selection with naive scFv libraries, it has turned out that six selection rounds were necessary to obtain specific binders (Hanes et al., 2000b). The designed ankyrin repeat protein libraries routinely yield binders after only three to four rounds (Binz et al., 2004). We suspect this might be due to the fact that these repeat proteins, which are extremely well expressed in E. coli, fold well, and are very stable, are also displayed better in vitro than scFv fragments, which are intrinsically somewhat more aggregation prone.
B. Affinity Maturation of Binders
In vitro evolution, the alternation of diversification and selection, is a powerful strategy used to improve proteins. Ribosome display is an ideal platform to perform such experiments. It allows very fast selection cycles and the PCR step is ideal to generate diversity in between the selection rounds. This diversification of the selected pools by random mutations increases the sampled sequence space. Error-prone PCR, e.g., using high Mn2+ concentrations (Leung et al., 1989), imbalanced dNTP concentrations (Cadwell and Joyce, 1994), or nucleotide analogues (Zaccolo and Gherardi, 1999; Zaccolo et al., 1996), is one strategy often applied to achieve diversification. Another powerful strategy to create diversity is DNA shuffling on the selected pools in between the rounds (Stemmer, 1994).
When compared to a selection experiment from a naïve library, the challenge in affinity maturation is different. The applied library will usually be created from a single clone or a pool of clones, which are already good binders. Since in affinity maturation we do not wish to select for binding as such but for better binding, we need an adjustable selection pressure (Jermutus et al., 2001). In principle, two strategies can be used to select tight binders out of a pool of binders.
The first affinity maturation strategy is to supply very little of the immobilized antigen. The concept is that the binders all compete with each other and, at equilibrium, the tight binders keep the binding spaces occupied. In practice, however, there are several complications to this concept. At extremely low target molecule concentrations, the relative proportion of unspecific binding sites gets very high (BSA, neutravidin) so that great care has to be taken to avoid unspecific binders. Also, if medium-affinity binders outnumber the tight binders, the enrichment will be very slow. Finally, if binding is very tight, the equilibrium is reached exceedingly slowly.
The second very successful strategy for affinity maturation is off-rate selection. The assumption fundamental to off-rate selection is that the on-rate of most protein-protein interactions is in the range of 105-106 M-1s-1 (Wodak and Janin, 2002) and proteinligand interactions in the range of 106-107 M-1s-1, such that the affinity is largely governed by the off-rate. By first incubating the ribosome-displayed polypeptide with the biotinylated target molecule (typically for 1 h) followed by the addition of a large excess of nonbiotinylated target (1000-fold excess), the selection pressure is governed by the dissociation of the binders from the biotinylated target. While tight binders will remain bound to the biotinylated target, others will dissociate and then rebind to the excess of unbiotinylated target. The incubation time equals the selection pressure and should be adjusted to the expected offrate. If the recovery is poor, it is often useful to include a nonselective round to enrich the binders.
C. Evolution of Properties Other Than High- Affinity Binding
The selection strategies for molecular properties other than high-affinity binding were summarised in Section I. A library can be evolved for these properties, just as it can be for affinity. It exceeds the scope of this chapter to discuss each strategy in detail, and there are many more possibilities that have not been explored experimentally.
The authors thank all former and present members of the Pliickthun laboratory involved in ribosome display who helped in developing the present protocol.
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