Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

Enhancing the affinity of SEB-binding peptides by repeating their sequence

The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12-mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage-display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB-binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12-mer peptide sequences. A 10-fold increase was observed in the binding affinity of one of the two-repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems.     

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities K_D∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.     

In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo

One approach to targeted therapies for cardiovascular disease relies on isolating ligands that enhance the tissue-specific uptake of genes or drugs by heart cells. To obtain heart-targeting ligands, phage display biopanning was used to isolate a 20-mer peptide that binds to isolated primary cardiomyocytes. The isolated phage, PCM.1, displays the peptide WLSEAGPVVTVRALRGTGSW, and binds these cells 180 times better than a control phage from the library. Furthermore, phage displaying this peptide preferentially bind to cardiomyocytes when compared with a panel of other cell types. A BLAST search revealed that this peptide contains a 12 amino acid segment with sequence identity to a peptide in tenascin-X, an extracellular matrix protein. Synthetic peptides containing the complete 20-mer or a 12-mer tenascin peptide partially blocked phage binding to the cardiomyocytes. We developed a quantitative real-time PCR assay to assess uptake of this phage by tissues in vivo. Using this assay, preferential localization of the PCM.1 phage in heart was observed compared to the uptake of this phage by other tissues or other phage by heart. Furthermore, PCM.1 phage was associated with cardiomyocytes isolated from mice treated with a phage in vivo. These results demonstrate the utility of biopanning on isolated cells for identifying specific binding peptides that can target a tissue in vivo.     

Discovery of high-affinity peptide binders to BLyS by phage display

B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.     

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

RELIC--a bioinformatics server for combinatorial peptide analysis and identification of protein-ligand interaction sites

Phage display technology provides a versatile tool for exploring the interactions between proteins, peptides and small molecule ligands. Quantitative analysis of peptide population sequence diversity and bias patterns has the power to significantly enhance the impact of these methods [1, 2]. We have developed a suite of computational tools for the analysis of peptide populations and made them accessible by integrating fifteen software programs for the analysis of combinatorial peptide sequences into the REceptor LIgand Contacts (RELIC) relational database and web-server. These programs have been developed for the analysis of statistical properties of peptide populations; identification of weak consensus sequences within these populations; and the comparison of these peptide sequences to those of naturally occurring proteins. RELIC is particularly suited to the analysis of peptide populations affinity selected with a small molecule ligand such as a drug or metabolite. Within this functional context, the ability to identify potential small molecule binding proteins using combinatorial peptide screening will accelerate as more ligands are screened and more genome sequences become available. The broader impact of this work is the addition of a novel means of analyzing peptide populations to the phage display community.     

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.     

Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries.

Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.     

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.     

Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

Identification and rational redesign of peptide ligands to CRIP1, a novel biomarker for cancers

Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10-28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides.     

Identification of a novel peptide ligand of human vascular endothelia growth factor receptor 3 for targeted tumour diagnosis and therapy

Human vascular endothelia growth factor receptor 3 (VEGFR-3) is up-regulated in a variety of human cancers. It is a potentially rational target for drug delivery. To identify novel ligands with specific binding capabilities to VEGFR-3, we screened a phage display peptide library and found a consensus motif of the peptides which is displayed by the positive phages CSDxxHxWC (x is any amino acid). The phage displaying peptide CSDSWHYWC (designated as P1) exhibited the highest affinity to VEGFR-3 in phage ELISA and the chemically synthesized P1 could bind to VEGFR-3 specifically in a dose-dependent manner. In addition, the flow cytometry assay and immunofluorescence showed that the FITC labelled P1 could bind to VEGFR-3 positive carcinoma cells with specificity. Our study suggests that P1 may be a homing peptide for treatment of tumours.     

Panning of a Phage Display Library against a Synthetic Capsule for Peptide Ligands That Bind to the Native Capsule of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.     

Study of Peptide Mimetics of Hepatitis A Virus Conjugated to Keyhole Limpet Hemocyanin and as Multiple Antigen Peptide System

Mimotopes mimic binding properties of natural antigen epitopes. They could be used for vaccine design, drugs development, and diagnostic assays. We have previously identified four bacteriophages displaying hepatitis A virus (HAV) mimotopes from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Three of these HAV mimotopes showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV and the four induced specific anti-HAV antibodies. In the present work, four conjugations were done. In each of them, a linear peptide (46, 53, 54 or 56) containing the amino-acid sequence of the corresponding mimotope was conjugated to keyhole limpet hemocyanin (KLH). Conjugation products were named: 46KLH, 53KLH, 54KLH and 56KLH. A two-arm multiple antigen peptide (MAP) system containing peptide sequence 46, and a second MAP containing two copies of peptide sequence 56 were synthesized and dimerized, to obtain the heterodimeric four-arms MAP (named MAP46-56) containing two copies of peptides 46 and 56. Mice were immunized with peptides conjugated to KLH and MAP46-56 to evaluate the ability of these two forms of mimotope presentation, to elicit antibodies that bind to the original antigen. KLH conjugated peptides rendered the highest levels of anti-peptide antibodies and were the only ones that induced specific anti-HAV antibodies. The results of immunizations showed that for the mimotopes chosen here, conjugation to a carrier protein was the most effective option to induce antibodies that cross-reacted with the natural antigen.