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At least 19 recordsLinked to original sources
PubMed1996

Identification of the peptides that stimulate the phosphoinositide hydrolysis in lymphocyte cell lines from peptide libraries.

Peptides which stimulate the formation of inositol phosphates (InoPs) in lymphocyte cell lines were identified by screening synthetic peptide libraries composed of random sequences of hexapeptides. The peptides containing the consensus sequence XKYX(P/V)M were found to be most active in the phospholipase C (PLC)-mediated formation of InoPs in a human B myeloma cell line, U266. The peptides also stimulated the phosphoinositide hydrolysis and the release of [Ca2+]i in HL60 and U937 cell lines. On the other hand, these peptides showed no effect in the following cell lines: NIH3T3, PC12, Daudi, Sp2, Jurkat, H9, Molt-4, SupT-1, K562, and RBL-2H3. The result suggests the possibility that the peptides may have cell type specificity. Experiments with one of the active peptides, WKYMVM-NH2 showed that its action mimics the effect of AlF4- which is a G-protein activator in the InoPs generation, and pertussis toxin partially blocked the InoPs accumulation and [Ca2+]i release induced by the peptide in the U266 cells. Binding assays with the peptide labeled with 125I showed that U266 cells have a saturable number of binding sites for the peptide. Taken together, these results suggest that the peptides could activate PLC-mediated signal transduction via a pertussis toxin-sensitive G-protein coupled receptor in certain cell types.

Amino Acid Sequence↗
PubMed1992

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.

Amino Acid Sequence↗
PubMedDate not supplied

Multipin technology in the preparation and screening of peptide libraries.

Peptide libraries are relatively new sources of enormous numbers of unique compounds, fodder for the mill of drug discovery programs. Their enormous diversity derives from vast numbers of combinations of a small number of monomers (Geysen et al., 1986). For example, a complete hexapeptide library synthesized from just 10 monomers (amino acids) has one million unique compounds in it. In principle, other types of combinatorial libraries can have equally vast numbers of members; for example, the monomers can be N-acyl glycines, giving rise to the so-called "peptoids" (Simon et al., 1992); or the monomers could be monosaccharides or nucleotides.

Biotechnology↗
PubMed1996

Elucidation of the core residues of an epitope using membrane-based combinatorial peptide libraries.

Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid. The libraries were probed with the antibody 201/9, and the bound antibody was detected with a sensitive chemiluminescent method. In the first cycle, the peptide mixtures X-Phe-X-Gln-X-X-X-X showed the strongest signal development. In the second cycle Phe and Gln were incorporated into new libraries consisting of sequences O1-Phe-X-Gln-X-X-X-X, X-Phe-O3-Gln-X-X-X-X, X-Phe-X-Gln-O5-X-X-X, X-Phe-X-Gln-X-O6-X-X, X-Phe-X-Gln-X-X-O7-X, and X-Phe-X-Gln-X-X-X-O8. After probing these new peptides, the residues representing the core sequence of the epitope for monoclonal antibody 201/9 were elucidated. The sequence Ser-Phe-Leu-Gln-Glu-Asn, identified as the immunodominant epitope, correlates well with the sequence Ser-Phe-Leu-Gln-Glu-Ala previously identified (Gao, B., and Esnouf, M. P. (1996) J. Immunol. 157, 183-188) in a scan of overlapping peptides based on the sequence of human beta-factor XIIa.

Amino Acid Sequence↗
PubMed1994

Major histocompatibility complex class I allele-specific peptide libraries: identification of peptides that mimic an H-Y T cell epitope.

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.

Amino Acid Sequence↗
PubMed1996

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Animals↗
PubMed1994

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Amino Acid Sequence↗
PubMed1995

Use of a constrain phage displayed-peptide library for the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7).

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.

Amino Acid Sequence↗
PubMed1996

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries.

Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10(7) hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.

Amino Acid Sequence↗
PubMed1995

Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.

Amino Acid Sequence↗
PubMed1995

Novel avidin and streptavidin binding sequences found in synthetic peptide libraries.

Synthetic resin-bound peptide libraries made of protein L-amino acids have been synthesized. During screening of the libraries, peptides that bind to avidin have been identified containing a novel motif with two histidines separated by one residue. A sub-library was synthesized and screened, and new critical residues appeared surrounding the two histidines. Additionally peptide libraries made of D-amino acids have been screened with avidin and streptavidin and novel motifs have been found.

Amino Acid Sequence↗
PubMed1995

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Amino Acid Sequence↗
PubMed1994

An in vitro polysome display system for identifying ligands from very large peptide libraries.

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.

Amino Acid Sequence↗
PubMed1995

Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library.

A bacteriophage peptide library containing a random 15-amino-acid insert was screened for identification of peptide sequence(s) that bind pp60(c-src). Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60(c-src) tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a Ki of 24 microM, whereas replacing the glycines with proline increased the Ki value to 3.1 mM.

Amino Acid Sequence↗
PubMed1996

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗