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PubMed1995

Protein ligands of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers.

A filamentous phage-displayed random hexapeptide library was screened on the adenovirus type 2 (Ad2) penton capsomer and its separate domains, penton base, full-length fiber, fiber shaft and fiber knob. Affinity supports were designed to immobilize the penton ligate with a preferred orientation, via immuno-adsorption to pre-coated antibody. Three classes of phagotopes were distinguished in the eluates from the penton and fiber domains. (i) The first class represented peptide sequences identified in certain Ad2 capsid proteins, protein IIIa, protein pVIII, penton base and penton fiber. Data from specific ligand elution of phages bound to fiber and penton base wild-types and mutants suggested that the region overlapping the RLSNLLG motif at residues 254-260 in the penton base and the FNPVYP motif at residues 11-16 in the fiber tail formed mutual interacting sites in the penton capsomer. (ii) The second class consisted of phagotopes homologous to peptide sequences found in host cell membrane proteins involved in receptor or adhesion functions. One of the most abundant species corresponded to a conserved motif present in the beta-strand B of type III modules of human fibronectin. In addition, phages which were screened for their failure to bind to penton base RGD mutants were found to carry consensus motifs to peptide sequences present in the RGD recognition site of human integrin beta subunits. (iii) The third class comprised peptide motifs common to both viral and cellular proteins, suggesting that a mechanism of ligand exchange could occur during virus entry and uncoating, and virus assembly and release.

Adenoviruses, Human↗

PubMedDate not supplied

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance.

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.

Animals↗

PubMed1995

Screening a monoclonal antibody with a fusion-phage display library shows a discontinuity in a linear epitope within PreS1 of hepatitis B virus.

The epitope recognized by the monoclonal antibody MA18/7, specific for the PreS1-domain of the hepatitis B virus surface antigen, has been defined precisely by means of a library of fusion-phage carrying random hexapeptides on the tip of filamentous phage fd particles. Phage, isolated after only one round of affinity selection, displayed hexapeptides showing strong conservation of the PreS1 primary sequence in the region 19-23 with three noncontiguous residues, DP (20 and 21) and F (23) appearing in phage that bound the antibody. The importance of these core residues was supported by comparing the antibody binding of individual phage in solution, which provided relative dissociation constants for these interactions. Replacement of F (23) by Y was the only substitution observed in the three core residues, and resulted in somewhat weaker binding. Synthetic tetra- and hexapeptides containing these key residues inhibited the reaction between the phage and the antibody.

Amino Acid Sequence↗

PubMed2005

Design, construction, and characterization of a large synthetic human antibody phage display library.

Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.

Antibodies, Monoclonal↗

PubMed1993

Identification of the amino acids comprising a surface-exposed epitope within the nucleotide-binding domain of the Na+,K(+)-ATPase using a random peptide library.

Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8-P1-A3 has been shown to bind to the catalytic (alpha) subunit of the Na+,K(+)-ATPase holoenzyme and the synthetic peptide sequence 496-HLLVMK*GAPER-506, which includes Lys 501 (K*), the major site for fluorescein-5'-isothiocyanate labeling of the Na+,K(+)-ATPase. This sequence region of alpha is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W. R. & Green, N. W., 1989, Eur. J. Biochem. 179, 241-248). In this study we have determined M8-P1-A3's ability to recognize the alpha-subunit or homologous E1E2-ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or "epitope" library, recently developed by Scott and Smith (1990, Science 249, 386-390) and Devlin et al. (Devlin, J. J., Panganiban, L. C., & Devlin, P. E., 1990, Science 249, 404-406), has served as a convenient technique to confirm the species-specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8-P1-A3 epitope was found to consist of the five amino acid 494-PRHLL-498 sequence stretch of alpha, with residues PRxLx being critical for antibody recognition.

Adenosine Triphosphate↗

PubMed1993

Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

Amino Acid Sequence↗

PubMed1994

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

Antibodies, Monoclonal↗

PubMed1996

Selection of trypsin inhibitors in phage peptide library.

The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest. We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library. After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported. All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.

Amino Acid Sequence↗

PubMed1999

Toward selection of internalizing antibodies from phage libraries.

Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).

Antibodies, Bispecific↗

PubMed2001

A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages.

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.

Avidin↗

PubMed1993

fd coat protein structure in membrane environments.

The membrane bound form of bacteriophage fd coat protein has a long hydrophobic membrane spanning helix and a shorter amphipathic helix in the plane of the bilayer. Residues near the N and C termini and in the turn connecting the two helices are mobile. The locations and orientations of the helical secondary structure elements and the protein backbone dynamics were characterized by combining results from multidimensional solution NMR experiments on protein samples in micelles and high resolution solid-state NMR experiments on protein samples in oriented and unoriented lipid bilayers. The coat protein is a monomer in micelles. The secondary structure of the membrane bound form of fd coat protein is very similar to that of the structural form found in the virus particles, since it is nearly all alpha helix. However, the membrane bound form of the protein differs from the structural form of the protein in virus particles in the arrangement of the secondary structure, since the membrane bound form of the protein has two distinct helical domains oriented perpendicular to each other and the structural form of the protein in the virus particles has a nearly continuous helix aligned approximately along the filament axis. In addition, there are substantial differences in the dynamics of residues in the bend between the two helices and near the C terminus, since they are mobile in the membrane bound form of the protein and not in the virus particles. Residues 1 to 5 at the N terminus are highly mobile and unstructured in both the membrane bound and structural forms of the coat protein.

Amides↗

PubMed1995

Matching electrostatic charge between DNA and coat protein in filamentous bacteriophage. Fibre diffraction of charge-deletion mutants.

The virion of Ff (fd, f1, M13) filamentous bacteriophage consists of a long tube of coat protein subunits in a shingled, helical array, surrounding a genome of circular single-stranded DNA. Modified fd virions have been generated by a mutation (K48A) that removes one positive charge from each coat protein subunit in the C-terminal region of the polypeptide chain facing the DNA. The number of nucleotides in the mutant DNA is unchanged, but the K48A virions are 35% longer than wild-type. We have measured the X-ray diffraction attributable to single virions in hydrated gels of wild-type and K48A bacteriophages. Most of the diffraction pattern shows no significant difference between wild-type and K48A. Since the DNA is only about 12% by weight of the wild-type virion, the diffraction pattern is dominated by the protein contribution, and the absence of significant differences indicates that there are no significant changes in the symmetry or structure of the protein coat. But there is a change in the diffraction pattern in a region where the DNA and protein contributions are comparable. The diffraction pattern of the K48A mutant shows an increase in intensity of one of the weaker equatorial peaks, relative to wild-type, in a region where the protein contribution has negative sign but the DNA contribution has positive sign. This is consistent with a decrease in the ratio of DNA:protein per unit length of the K48A mutant. The results support the view that the protein forms a sheath lined with positive charges interacting electrostatically and non-specifically with a negatively charged DNA core of matching charge density. The lower positive charge density lining the capsid in the K48A mutant means that correspondingly fewer nucleotides can be packaged per coat protein subunit, which in turn requires an elongation of the DNA inside the virion. A longer virion is thus required to package the same amount of DNA. Within the error of measurement, the number of positive charges on the protein interacting with the DNA is the same in K48A as in the wild-type, despite the fact that the mutant is 35% longer than the wild-type.

Capsid↗

PubMed1994

In vivo characterization of mutants of the bacteriophage f1 gene V protein isolated by saturation mutagenesis.

The gene V protein of bacteriophage f1 binds to single-stranded nucleic acids and is essential for propagation of phage f1. We tested the function of gene V protein mutants with single amino acid substitutions in two ways: by the ability of the mutant proteins to support phage growth, and by the ability of the mutant proteins, when expressed at high levels, to inhibit the growth of Escherichia coli. The results of the tests were used to identify sites in the protein that are relatively tolerant or intolerant to substitution, where tolerant sites are defined as those where most substitutions do not affect the function of the protein. The two assays generally yielded similar results for the tolerance of sites to substitution. Many sites that are less than 10% exposed to the solvent are relatively intolerant of substitution, with even very conservative substitutions leading to loss of function in some cases such as Ile to Leu at residue 6. Some buried sites such as Ile47 are more tolerant, with even a substitution of Ile to Thr leading to a functional protein based on the ability of the proteins to inhibit the growth of E. coli. Some surface sites in the protein (> 10% exposure to solvent) that are thought to be near the location of bound oligonucleotides, such as Arg16, Val19, Ser20, Arg21, Tyr26, Lys46 and Arg80, are sensitive to substitution. Other side-chains thought to be close to bound oligonucleotides, including Leu28, can be replaced with a number of amino acids with little loss of function based on either assay. Most non-Gly/Pro surface residues thought to be distant from the locations of bound oligonucleotides are relatively tolerant of substitution, except for two small residues (Ala11 and Thr14), two aromatic residues (Tyr34 and Tyr56), two residues that are only partially exposed to solvent (Asn29 and Val70), and three residues that have been proposed to be at the dimer-dimer interface formed when gene V protein binds to nucleic acids (Glu40, Tyr41 and Arg82).

DNA, Single-Stranded↗

PubMed1994

Selection of beta-lactamase on filamentous bacteriophage by catalytic activity.

Recently the display of repertoires of peptides and proteins on the surface of filamentous phage, and selection of the phage by binding to a ligand, has allowed the isolation of peptides and proteins with rare binding activities. Furthermore, phages displaying enzymes (phage enzymes) have been selected by affinity of binding to inhibitors. Here we show, using a suicide inhibitor, that phage enzymes can also be selected by their catalytic activity. Two phage enzymes were constructed by fusion to the minor coat protein of the phage (g3p), displaying either an active beta-lactamase or a catalytically inactive mutant in which the essential serine of the active site was mutated to alanine. The phages were then incubated with a beta-lactamase suicide inhibitor connected by a spacer to a biotin moiety. The active (but not the inactive) phages were labelled, and the active phages selected from mixtures with inactive phages by binding and elution from streptavidin-coated beads. The selection ratio for active versus inactive phages (about ten on elution of the phages by reduction of an S-S bond in the spacer between the warhead and biotin) could be improved to about 50 on elution by proteolytic cleavage of beta-lactamase from g3p at an intervening factor X site. Selection of phage-enzymes by catalysis may provide a means of creating new enzymes and refining their catalytic properties.

Alanine↗

PubMed1994

pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.

Filamentous phage pIV is an outer membrane protein required for phage assembly and secretion. Chemical cross-linking and sedimentation experiments have been used to demonstrate that pIV from f1-infected Escherichia coli exists as a homo-multimer, probably composed of 10 to 12 subunits. pIV secreted from spheroplasts remains soluble and does not form multimers. Synthesis of pIV from distantly related filamentous phages or from a bacterial homolog that participates in a specialized form of extra-cellular protein secretion in the same cell with pIVf1 resulted in the formation of mixed multimers. This suggests that the homologous proteins themselves form homo-multimers. These structures could form gated channels that conduct assembling phage or specific substrate proteins across the outer membrane to the extracellular milieu.

Bacterial Proteins↗

PubMed1994

Structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. The V3 loop of HIV-1 gp120.

The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is an intra-chain disulphide-bridged loop, designated V3, in the third hypervariable region of the surface glycoprotein gp120. Peptide sequences from the V3 loop of gp120 from HIV-1 strain MN (HIV-1MN) were engineered into the N-terminal region of the major coat protein of filamentous bacteriophage fd, leading to their display in multiple copies on the surface of the bacteriophage virion. Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope. They were recognised by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus. In addition, antibody production could be stimulated by simultaneous inoculation with T-cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins and is a promising vaccine model.

Amino Acid Sequence↗

PubMed1995

Modifying filamentous phage capsid: limits in the size of the major capsid protein.

Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.

Amino Acid Sequence↗

PubMed1995

Structures of fd gene 5 protein.nucleic acid complexes: a combined solution scattering and electron microscopy study.

Small-angle scattering and electron microscopy studies of fd gene 5 protein (g5p) and reconstituted g5p.nucleic acid complexes have been used to test models for the complexes and evaluate their uniqueness. In addition, we have obtained new information on the dependence of nucleotide type and protein/nucleotide (P/N) ratio on the structure of the complexes. Reconstituted complexes were made with single-stranded fd viral DNA (fd ssDNA), poly[d(A)] and poly[r(A)]. All complexes form similar left-handed, flexible superhelices having approximately the same diameter, but the pitch differs among these complexes. The g5p protein is a dimer in solution and the dimers associate to form a superhelical framework to which the polynucleotide is attached. The combined X-ray and neutron scattering data confirm the nucleic acid is inside the protein superhelix. A Monte Carlo integration modeling procedure applied to the scattering data was used to systematically test large numbers of possible models for each complex, and previously proposed models based on parameters obtained from electron microscopy were found to be essentially correct and unique. The data on the complexes with different P/N ratios showed that mass per unit length values decreased while the rise per dimer and pitch of the superhelix increased for g5p.fd-ssDNA complexes with decreasing P/N ratios.

DNA, Single-Stranded↗