Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Peptide Library”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed1999

Calcineurin B- and calmodulin-binding preferences identified with phage-displayed peptide libraries.

Calcineurin B (CnB) and calmodulin (CaM) are two structurally similar but functionally distinct 'EF-hand' Ca2+-binding proteins. CnB is the regulatory subunit of the CaM-stimulated protein phosphatase, calcineurin. CaM is a unique multifunctional protein that interacts with and modulates the activity of many target proteins. CnB and CaM are both required for the full activation of the phosphatase activity of calcineurin and are not interchangeable. The two proteins recognize distinct binding sites on calcineurin A subunit (CnA) and perform different functions. Phage-displayed peptide libraries (pIII and pVIII libraries) were screened with CnB and CaM to isolate peptides that could then be compared to determine if there were binding preferences of the two proteins. The Ca2+-dependent binding of phage-displayed peptides to CnB and CaM is specifically blocked by synthetic peptides derived from the CnB-binding domain of CnA and the CaM-binding domain of myosin light chain kinase respectively. Both CnB- and CaM-binding peptides have a high content of tryptophan and leucine, but CnB-binding peptides are more hydrophobic than CaM-binding peptides. CnB-binding peptides are negatively charged with clusters of hydrophobic residues rich in phenylalanine, whereas the CaM-binding peptides are positively charged and often contain an Arg/Lys-Trp motif. The binding preferences identified with peptide libraries are consistent with the features of the CnB-binding domains of all CnA isoforms and the CaM-binding domains of CaM targets.

Amino Acid Sequence↗
PubMed1996

Identification of HLA-DR9 (DRB1*0901)-binding peptide motifs using a phage fUSE5 random peptide library.

We identified HLA-DRB1*0901-binding peptides by affinity-based selection of a phage random peptide library using the biotinylated DR9 complex. Analogue peptides with single amino acid residue substitutions of a DR9 binder revealed that two major anchors (WxxS, where x is any amino acid) play an essential role in binding to DR9. Determination of the binding affinity of synthetic wild-type-based analogue peptides showed that substituting W to F or L, and S to A, V, or F allow high affinity binding with DR9. Collectively, DR9-binding peptide motifs identified in this study are characteristic in that (a) only two anchors of the NH2-terminal half of binding peptides play important roles in binding, and (b) small neutral hydrophilic Ser is allowed as the second anchor for high-affinity binding, unlike the other DR-binding motifs heretofore reported. The implications of our results are discussed in light of the HLA-DR9-associated susceptibility to juvenile-onset myasthenia gravis and systemic lupus erythematosus with antiphospholipid syndrome, in particular, T-cell responses to autoantigens.

Amino Acid Sequence↗
PubMed2001

Functional screening of a retroviral peptide library for MHC class I presentation.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries.

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.

3T3 Cells↗
PubMed2004

Identification of HLA-Cw6.02 and HLA-Cw7.01 allele-specific binding motifs by screening synthetic peptide libraries.

Unlike HLA-A and HLA-B, few peptide epitope motifs have been reported for HLA-C molecules. However, a number of cytotoxic T-lymphocyte epitopes derived from tumor antigens that bind to HLA-C molecules have been described. Here we report peptide-binding motifs for both HLA-Cw6.02 and HLA-Cw7.01 molecules. Recombinant human HLA molecules were generated and used to screen combinatorial 9mer peptide libraries. Complexes of HLA molecules properly folded and associated with beta2-microglobulin and peptides were identified using a conformation-specific HLA class I antibody conjugated to alkaline phosphatase. In the presence of substrate, peptide beads can be readily isolated and microsequenced to determine peptide identity. Of the peptides that bound to HLA-Cw6.02 and HLA-Cw7.01, 19 and 18 peptides, respectively, were sequenced, allowing motif identification for each C allele. This is the first report of an HLA-Cw7.01 peptide motif and extends the findings of Falk et al. [(1993) Proc Natl Acad Sci USA 90:12005] for an HLA-Cw6.02 motif. Anchoring amino acids for the HLA-Cw6.02 motif were phenylalanine or tyrosine in position (P)1, arginine in P2, and an aliphatic/aromatic residue at P9. Anchoring residues for HLA-Cw7.01 were positively charged amino acids in P1 and P2. Unlike most other HLA molecules, we were unable to assign P9 an anchoring residue, and we suspect that HLA-Cw7.01 binds peptides in an unconventional manner. Additionally, preferred amino acids were identified for both molecules. Identification of HLA-Cw6.02 and HLA-Cw7.01 peptide-binding motifs makes a significant contribution to the C allele peptide-binding motifs and will allow investigators to predict, design, and test HLA-Cw6.02 and HLA-Cw7.01 engineered peptides for immunotherapy.

Alkaline Phosphatase↗
PubMed1999

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Algorithms↗
PubMed2006

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library.

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Amino Acid Sequence↗
PubMed2004

A specific heptapeptide from a phage display peptide library homes to bone marrow and binds to primitive hematopoietic stem cells.

Phage display peptide libraries have enabled the discovery of peptides that selectively target specific organs. Selection of organ-specific peptides is mediated through binding of peptides displayed on phage coat protein to adhesion molecules expressed within targeted organs. Hematopoietic stem cells selectively home to bone marrow, and certain adhesion receptors critical to this function have been demonstrated. Using a phage display library, we identified a specific peptide that trafficked to murine bone marrow in vivo. We independently isolated exactly the same heptapeptide from the entire library by in vitro biopanning on primitive lineage-depleted, Hoechst 33342(dull)/rhodamine 123(dull) murine bone marrow stem cells and confirmed peptide binding to these cells by immunofluorescence studies. We demonstrated bone marrow-specific homing of the peptide by an in vivo assay in which the animals were injected with the phage displaying peptide sequence, and immunofluorescence analysis of multiple organs was performed. We also showed that the peptide significantly decreased the homing of stem cells to the bone marrow but not to the spleen 3 hours after transplantation using fluorescently labeled Lin(-)Sca(+) hematopoietic cells in an in vivo homing assay. The peptide sequence has a partial (5/7) amino acid sequence homology with a region of CD84. This discovery represents the first application of the phage display methodology to the bone marrow and stem cells and led to the identification of a specific heptapeptide that homes to bone marrow, binds to primitive stem cells, and plays a role in stem cell homing.

Animals↗
PubMed1996

Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library.

A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a "one bead, one peptide" combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P2, P1 and P'2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with Ki values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Antibody epitopes probed by immunoselected phage-display library peptides in members of a family with various rheumatic manifestations.

OBJECTIVE: The random peptide combinatorial phage library approach overcomes the problem of lack of structural information about the aetiological agent or the antigen responsible for a given disease. Here, we used such a strategy to gain insight into the aetiology of rheumatoid arthritis (RA). METHODS: We analyzed the reactivity of serum antibodies from a family with various rheumatic manifestations against RA-immunoselected nanopeptides displayed on phage particles. RESULTS AND CONCLUSION: We found that within the same family, there was a difference in antibody reactivity against the peptides tested. The IgG isotype of the peptide reactive antibodies indicated that the observed reactivities were not related to the presence of polyreactive IgM antibodies. Furthermore, it is unlikely that the observed reactivity was due to rheumatoid factors (RF), since two patients who were positive for the immunoselected Pep3 peptide (LSSREPQAR) were RF negative. We also found that the serum of one patient with polyarthralgias also reacted with the same peptide bound by the RA serum, which may suggest the implication of a common aetiological agent in the apparition of this antibody reactivity. Finally, we noted that one patient with Sjögren's syndrome had antibodies to the RA peptide, which may indicate a potential relationship between these two autoimmune diseases.

Arthritis, Rheumatoid↗
PubMed2003

Generation of microglia specific reagents from phage displayed peptide libraries.

The present report concerns the generation of specific markers and the establishment of a selection procedure for microglia specific molecules from phage displayed peptide libraries. Negative selection against a mouse monocytic cell line (IC-21) and positive selection against primary mouse microglia was combined in the selection procedures using a mixture of two random peptide libraries displayed on phage. In a first set of experiments, one clone was selected that bound microglia and IC-21 cells to equal extent, and three clones that bound to unsorted primary microglia to substantially higher levels than to IC-21 cells. In the second series of experiments, microglia and IC-21 cells were mixed and CD45-positive microglia cells were collected using a FACS sorter. From the latter selection series, three clones were found that preferentially bound to microglia cells. The binding of one of the six selected microglia specific phage clones, clone V-1:19, was competed/inhibited in experiments using soluble synthetic peptides corresponding to the binding motif of the phage clone. The specific inhibition to microglia cells by this synthetic peptide was effective in the concentration range of 0.5-20 microM. The preferential binding of clone V-1:19 to microglia like cells was further demonstrated by staining a panel of cell lines and purified primary mouse microglia.

Amino Acid Sequence↗
PubMed2003

Phage display of random peptide libraries: applications, limits, and potential.

The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.

Animals↗
PubMed2006

SIRT1 top 40 hits: use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity.

A novel, high-throughput method for determining deacetylase substrate specificity was developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central epsilon-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry. In-solution deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a context-dependent fashion.

Acetylation↗
PubMedDate not supplied

A combinatorial peptide library around variation of the human immunodeficiency virus (HIV-1) V3 domain leads to distinct T helper cell responses.

The hypervariable domain of the HIV gp120, the V3 loop domain, represents a target for neutralizing antibodies and for HIV vaccine strategies. In this study, we have investigated in murine species the potential cross-reactivity of immune responses elicited by immunization either with individual V3 peptides, derived from distinct HIV sequences (BRU, RF, SF2, MN and ELI sequences), or with a V3 combinatorial peptide library. We observed that individual V3 peptides are immunogenic but elicit a specific B- and T-cell immune response that is mainly restricted to the sequence of the immunizing peptide. In particular, T-cell responses that depend on T-cell receptor recognition of peptides bound to the molecules encoded by the major histocompatibility complex were significantly influenced by small differences in the peptide amino acid sequence. The combinatorial V3 peptide library, previously described as B- and T-cell immunogens, induced a more broadly reactive immune response, specially when T-cell cytokine secretion was used as a readout for restimulation of T-cells with individual V3 peptides. These data suggest that amino acid variations in the sequence of an antigenic peptide could lead to the induction of different transducing signals in the primed T-cell population and to the activation of T-cells with distinct cytokine secretion properties. These observations may have implications in the understanding of antigenic variability and in the design of vaccine strategies.

Amino Acid Sequence↗
PubMed1994

In vitro selection from protein and peptide libraries.

In vitro selection from molecular libraries has rapidly come of age as a protein-engineering tool. Dramatic increases in protein affinity can be engineered using phage-display libraries, and specific antibodies can be selected directly from a single 'naïve' library of their genes. Repertoires of small molecules are a potentially valuable resource for drug discovery. Libraries of linear peptides provide ligands for proteins that recognize continuous epitopes, and low-affinity mimics of some small molecules, but generally do not contain mimics of large molecular interfaces. Switching to constrained peptide formats, and deploying more diverse, non-peptide chemical libraries, may bring greater success.

Animals↗
PubMed2005

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening.

BACKGROUND: The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. RESULTS: Using a combination of peptide library selection, phosphorylation of optimal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. CONCLUSION: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.

Amino Acid Sequence↗
PubMed2003

DNA display for in vitro selection of diverse peptide libraries.

We describe the use of a DNA display system for in vitro selection of peptide ligands from a large library of peptides displayed on their encoding DNAs. The method permits completely in vitro construction of a DNA-tagged peptide library by using a wheat germ in vitro transcription/translation system compartmentalized in water-in-oil emulsions. Starting with a library of 10(9)-10(10) random decapeptides, 21 different peptide ligands were isolated for monoclonal antibody anti-FLAG M2. DNA display selected more diverse peptides with a DYKXXD consensus motif than previously reported phage display systems. Binding and recovery rates of three peptides were significantly higher than those of the original FLAG peptide, implying that these peptides would be superior to the FLAG peptide for purification of tagged proteins. The simplicity of DNA display enables two selection rounds per day to be conducted. Further, DNA display can overcome the limitations of previous display technologies by avoiding the use of bacterial cells and RNA tags. Thus, DNA display is expected to be useful for rapid screening of a wide variety of peptide ligands for corresponding receptors.

Amino Acid Sequence↗
PubMed2005

A systematic approach for comprehensive T-cell epitope discovery using peptide libraries.

MOTIVATION: T-cell response to peptides bound on MHC Class I or Class II molecules is essential for immune recognition of pathogens. T-cells are activated by specific peptide epitopes that are determined within the antigen processing pathways and presented on the surface of other cells bound to MHC molecules. To determine which part of allergenic or pathogenic proteins can stimulate T-cells is important for the treatment of diseases. We sought to take advantage of the falling cost of synthetic, screening grade peptides, and devise a comprehensive, non-hypothesis-driven screen for T-cell epitopes. We were interested in the study of celiac disease (CD) and used the ELISPOT technique to perform a large number of T-cell assays. We therefore needed to compensate for the lack of statistical data analysis methods for ELISPOT assays. RESULTS: We describe a method to comprehensively screen for T-cell epitopes within a family or a group of proteins. We have implemented an algorithm to generate a set of unique short peptide sequences that incorporate all possible epitopes within a group of proteins. T-cell assays were performed in 96-well plates using the ELISPOT assay to screen for responses in CD patients against any epitopes in glutens. We describe a statistical model to fit the data and an Expectation Maximization algorithm to estimate the parameters of interest and analyze the resulting data. AVAILABILITY: Implementations of our algorithms in R or Perl are available at http://bioinf.wehi.edu.au/folders/immunol.

Algorithms↗