Enrichment Of Phages Research Articles

Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner.

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.

Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.

A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.

GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies

Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein–protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS M) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.

Preparation of single chain variable fragment of MG7mAb by phage display technology

To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.

Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries.

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.

Model systems to study the parameters determining the success of phage antibody selections on complex antigens

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target “antigen”. We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.

Selective Enrichment and High-throughput Screening of Phage Surface-displayed eDNA Libraries from Complex Allergenic Systems

Abstract: Phage surface display technology, first described in 1985, enables the construction of large combinatorial peptide and antibody libraries. The basic concept of linking the phenotype, expressed as gene product displayed on the phage surface, to its genetic information integrated into the phage genome, allows the survey of large libraries for the presence of specific clones using the discriminative power of affinity purification. The selection procedure involves the enrichment of phage by binding to an immobilized target molecule. As a consequence of the physical linkage between genotype and phenotype, sequencing the DNA of the inteyrated section of the phage genome can readily elucidate the amino acid sequence of a displayed gene product. Phage surface display technology has revolutionized our ability to select agonist and antagonist peptides, antibodies with desired specificities, and DNA-binding molecules. We have extended phage surface display to access eDNA libraries with this powerful screening technology based on affinity selection of desired clones. Here we discuss construction of eDNA libraries displayed on the phage surface, selective enrichment of clones and robotics-based high-throughput screening of enriched libraries.

Identification of Peptides, Selected by Phage Display Technology, That Inhibit von Willebrand Factor Binding to Collagen

A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti–von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 μg/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 ± 12.7 μmol/L and 34.8 ± 8.59 μmol/L (mean ± SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.

Identification of Peptides, Selected by Phage Display Technology, That Inhibit von Willebrand Factor Binding to Collagen

AbstractA repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti–von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 μg/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 ± 12.7 μmol/L and 34.8 ± 8.59 μmol/L (mean ± SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.

Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene.

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 Å from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X 4library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X 4including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.

Efficient selection of scFv antibody phage by adsorption to in situ expressed antigens in tissue sections

The present report describes the development and application of an efficient method for the direct adsorption/selection of antibody phage using antigens expressed in situ in cryostat tissue sections. In a model system, scFv phage directed towards an epitope on the GA733-2 epithelial glycoprotein expressed in colorectal carcinoma tissue could be specifically enriched up to 1500 fold in single-pass experiments and a million fold after three rounds of selection. Enrichment efficacy was directly proportional to the fraction of antigen positive area over the total area. Sufficient enrichment was achieved at an area fraction of less than four percent, thereby permitting the selection of antibodies to sub-populations of cells or to tissue sub-structures. The general usefulness of the method was demonstrated when a combinatorial scFv antibody phage library derived from melanoma immunized non-human primates was selected in tissue sections of metastatic melanoma. Individual scFv antibodies from enriched phage populations demonstrated different binding specificities, reflected in extracellular and cellular tissue staining patterns which included tumor cell surface reactivity. This method should be particularly useful for the identification of antigens which are only expressed during specific in vivo conditions, and overcomes a major limitation of currently used selection protocols.

A Novel Approach to Human Anti-HLA mABs Production: Use of Phage Display Libraries

ABSTRACT: The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DRβ chain. Western blot analysis indicated that the 7 scFvs react with the DR1 α/β-dimer but not with free α- or β- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display.

We report here on the identification of phosphopetide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand Shc. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.

A model system for detection and isolation of a tumor cell surface antigen using antibody phage display

To establish a screening procedure for tumor cell-surface reactive Fabs, we used a model antigen/antibody system including the epidermal growth factor receptor (EGF-R) and the anti-EGF-R monoclonal antibody 425. The 425 Fab was displayed on the surface of M13 filamentous phage. In a screening assay for 425 phage binding to tumor cell surfaces, biotinylated 425-phage bound specifically to EGF-R-positive A431 epidermoid carcinoma cells and not to K562 non-expressor erythroleukemia cells. With a model library, the sensitivity of phage enrichment by phage binding to cell surfaces was one 425-phage in 20 000 unrelated phages after 4 rounds of panning on A431 cells. In a phage tissue screening assay, 425-phage, but not unrelated phage, bound specifically to melanoma cells expressing EGF-R. Epitope and idiotope specificity of 425-phage was demonstrated in phage competition assays, using as targets A431 cells and anti-idiotypic antibodies to monoclonal antibody 425, respectively. Finally, the EGF-R protein was directly isolated from A431 cell extracts, using biotinylated 425-phage. The data obtained with the 425 model library system demonstrate the usefulness of antibody phage display for the rapid identification and isolation of tumor or other disease-related cell surface antigens. © 1997 Elsevier Science B.V. All rights reserved.

The Functional Display of Interleukin-2 on Filamentous Phage

We report the novel display of interleukin-2 (IL-2) and an IL-2 analog, D126, on the surface of filamentous bacteriophage using a phagemid vector system. A synthetic human IL-2 gene and its D126 analog were fused to the carboxyl-terminal domain of the gene III minor phage coat protein. Expression of IL-2 and D126 was verified by their reactivity with an IL-2-specific antibody. Biological response of IL-2 phage on murine CTLL-2 cells was comparable to that of recombinant soluble IL-2, while the D126 phage displayed a reduced biological response similar to that previously measured by soluble D126 protein. Biosensor surface plasmon resonance was employed to verify binding of the IL-2 and D126 phage to the IL-2 αβcc receptor complex. A 41-fold enrichment of IL-2 phage over R408 helper phage was demonstrated in biopanning affinity selection studies employing biotinylated αβcc receptor complex. These biopanning studies are the first reports of affinity selection of IL-2 phage and demonstrate a novel use for the αβcc receptor complex. Together, these studies confirm that the structural integrity of IL-2 and D126 is maintained when they are displayed as a gIIIp fusion protein on phage particles and provide the foundation for further selection studies employing IL-2 analog phage libraries.

Combinatorial antibodies against human malignant melanoma.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for > 7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10(8) phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a > 10(6)-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody.

Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.

Epitope identification by polyclonal antibody from phage-displayed random peptide library.

Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligates is an effective method to define epitopes of protein antigens. However, it is thought that polyclonal antibodies might also serve as promising ligates for screening. We illustrate this approach by using recombinant human lymphotoxin (rhLT) polyclonal antibody as a model. The procedure consists in (a) affinity purification of polyclonal antibody to obtain the "monospecific" antibody, (b) screening against a phage-displayed random peptide library using the affinity-purified antibody, (c) plating the enriched phage on agar plates, randomly picking clones, and selecting the positive ones by dot blotting, (d) DNA sequencing of the positive clones and conducting a homology search against the protein sequence databank, and (e) confirming the epitopes by chemical peptide synthesis. By employing this procedure, we identified a dominant epitope RQHPKM, located at residues 15-20 of the human lymphotoxin amino acid sequence. The usefulness of this general procedure is discussed.