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PubMed1995

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes.

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

Amino Acid Sequence↗
PubMed1993

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Adenosine Triphosphatases↗
PubMed1995

A general route to fingerprint analyses of peptide-antibody interactions using a clustered amino acid peptide library: comparison with a phage display library.

We provide a general route to fingerprint analyses of peptide-antibody interactions using a novel chemically synthesized peptide library. A combinatorial clustered amino acid peptide library XO1O2O3O4X (O = one of six amino acid clusters [APG], [DE], [HKR], [NQST], [FYW] and [ILVM]; X = randomized position) bound to a continuous cellulose membrane support was designed to overcome the problem of combinatorial explosion in the synthesis of peptide libraries. This library served as the starting point for the identification and detailed characterization of a TGF alpha peptide epitope recognized by the antibody Tab2. By analysing 1728 hexapeptide mixtures and 1600 single hexapeptides we identified a large number of structurally different high affinity Tab2 binding molecules. Our data provide a detailed picture of the structural basis of this antibody-peptide interaction. In addition to the detection of key amino acids involved in Tab2 binding we observed a high variability of Tab2 binding sequences supporting an induced fit mechanism in antibody-peptide recognition. In contrast, a phage display hexapeptide library led to the detection of only one dominant Tab2 binding peptide. The data obtained also demonstrate the influence of phage proteins on the interaction between the antibody and the displayed peptide. Comparing both approaches with regard to ease of handling and identified sequences, the chemical libraries are clearly favored to study antibody-peptide interactions.

Amino Acid Sequence↗
PubMed1993

Selection of human anti-hapten antibodies from semisynthetic libraries.

Semisynthetic human Fab libraries were constructed, displayed on the surface of filamentous phage and selected for binding to three hapten conjugates. A number of Fabs were isolated and characterized with respect to affinity and specificity. Fabs exhibited affinities of between 80 and 29 nM, as determined by surface plasmon resonance, for the conjugate on which they were selected. Conservation of Asp101 in the third heavy-chain complementarity determining region (HCDR3) appears to be important in the construction of synthetically diverse repertoires.

Amino Acid Sequence↗
PubMed1993

Defining critical residues in the epitope for a HIV-neutralizing monoclonal antibody using phage display and peptide array technologies.

A peptide display library [Scott and Smith, Science 249 (1990) 386-390] was constructed that expressed 1.5 x 10(8) unique 20-amino-acid (aa) peptides fused to the N-terminus of the pIII coat protein of filamentous phage fd. This phage display library (PDL-20) was prepared using a degenerate oligodeoxyribonucleotide designed to minimize bias towards most aa. Characterization of the PDL-20 showed that all aa were present at the expected frequency and that there was no positional bias. Screening of this library with a HIV-1 isotype MN envelope reactive monoclonal antibody (mAb 58.2) using two different panning procedures showed that the biopanning technique was sensitive to one phage in 10(8). Analysis of peptide sequences from panning the mAb identified a core antibody recognition sequence of four aa residues (GPGR) and two preferred flanking residues on either side. This epitope occurred at various locations within the random aa segment demonstrating an absence of positional or nearest neighbor effects. Parallel panning experiments using an array of 266 synthetic peptides identified an epitope similar to that defined by the phage display library.

Amino Acid Sequence↗
PubMed1993

Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production.

A cloning and expression system allowing display of functional cDNAs or other gene products on the surface of filamentous phage has been developed, exploiting the high-affinity interaction of the Jun and Fos leucine zippers. Gene jun was expressed from a lacZ promoter as a fusion protein with the viral coat protein, pIII, thereby being structurally incorporated into phage particles during infection with a helper phage. Using a second lacZ promoter of the phagemid, gene fos was co-expressed as an N-terminal fusion peptide to cDNA library gene products, so that the resulting Fos-fusion proteins could become associated with the Jun-decorated phage particles. To avoid interphage exchange of fos-cDNA fusion products, cysteines were engineered at the N- and C-termini of each of the leucine zippers, providing a covalent link of the cDNA gene product to the genetic instructions required for its production. Dissociation between phage and cDNA gene products was readily achieved using reducing agents. Phages displaying gene products covalently anchored on their surface via the modified leucine zippers can be selectively enriched 10(4)-10(6)-fold over nonspecific phages using antibodies. Thus, this cloning system allows rapid isolation of rare mRNA products from complex cDNA libraries by enrichment with appropriate ligands. This approach should allow the expression and cloning of dimeric proteins by cDNA shuffling.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries.

The display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities, including haptens, proteins and viral particles. We have examined the possibility of isolating nucleic acid-binding Fab by constructing a combinatorial library of phage displaying Fab derived from autoimmune (MRL/lpr) mice. Autoimmune mice were chosen because they contain antibodies (Ab) reactive against nuclear components, including DNA, RNA and protein complexes. The library was panned against single-stranded (ss) calf thymus (CT) DNA and the selected Fabs were analyzed further. Characterization of the nucleic acid-binding phage led to the identification of two kinds of Fab with quite different properties. One Fab bound with high affinity a variety of ssDNA molecules, as well as several model RNA substrates. This Fab has been affinity purified to greater than 95% and competition studies revealed a marked preference for binding to poly(dT). The second Fab showed a reduced binding to RNA ligands and a restricted number of ssDNA molecules. Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heavy (H) chain V region from the strong nucleic acid-binding Fab was derived from a VH gene that is used recurrently in autoantibodies. This VH domain was most similar to an anti-ssDNA autoimmune monoclonal antibody (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Functional phage display of ciliary neurotrophic factor.

We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the alpha-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF alpha-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF alpha-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.

Amino Acid Sequence↗
PubMed1994

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

Amino Acid Sequence↗
PubMed1994

Vectors with the fd replicon for in vivo cloning and analysis of genes.

We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.

Cloning, Molecular↗
PubMed1995

Characterization of an open reading frame involved in site-specific integration of filamentous phage Cf1t from Xanthomonas campestris pv. citri.

Cf1t is a single-stranded DNA filamentous phage; a 1.9-kb segment of DNA from Cf1t was found to be responsible for site-specific integration into Xanthomonas campestris pv. citri (XW47), in the absence of any Xanthomonas origin of replication. Deletion analysis and introduction of amber stop codons into this fragment from Cf1t revealed an open reading frame (ORF344) which was involved in the integration function. The predicted amino-acid sequence of ORF344 bears no homology with conserved sequences of the integrase family.

Amino Acid Sequence↗
PubMed1996

New vectors for peptide display on the surface of filamentous bacteriophage.

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.

Amino Acid Sequence↗
PubMedDate not supplied

The role of the adsorption complex in the termination of filamentous phage assembly.

The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly. Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producing all of the other minor coat proteins. The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration. The data suggest the presence of the adsorption complex in these membranes. Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well. The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages. Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage. From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly.

Adsorption↗
PubMed2006

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries.

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.

Amino Acid Sequence↗
PubMed2005

Neutralizing human anti-B-cell-activating factor of the TNF family (BAFF) scFv selected from phage antibody library.

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K(aff)) of 2.5 x 10(7)M(-1) without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF.

Amino Acid Sequence↗
PubMed2005

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII.

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.

Capsid Proteins↗
PubMed2006

Phage display in the study of infectious diseases.

Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.

Adhesins, Bacterial↗