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At least 271 records · Page 15Linked to original sources
PubMed2000

Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication.

Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China. Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity. A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III. By biopanning, the fused peptides were bound to the biotinylated GCHV. Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91. Three rounds of affinity selection enriched the pool of inhibiting peptides. Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected. The TCID(50) of GCHV was decreased over 10000x. Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH(2)-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH. A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro.

Animals↗
PubMed1999

Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus.

Random peptide libraries (RPLs) screening with IDDM sera has identified 5 disease-specific 'mimotopes' displayed on phage (phagotopes). We characterised one phagotope (CH1p), by raising a rabbit antibody against the peptide insert on phage, which was employed in immunohistochemistry, Western blotting and cDNA libraries screening. The CH1p mimotope was detected in somatostatin cells of human islets and experimentally raised anti-osteopontin antibodies or human sera positive for the phagotope, detected a similar subpopulation of islet cells. The screening of cDNA library identified a clone corresponding to human osteopontin. In summary, RPLs proved to be successful in the identification of a novel islet-related autoantigen (osteopontin), whose significance in disease remains to be established.

Animals↗
PubMed2004

A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2.

Trastuzumab, a humanized antibody to HER-2, has been shown to be effective in the treatment of breast cancer in which HER-2 overexpression and metastasis occurs. In our search for an effective mimic epitope of HER-2 binding with trastuzumab and to develop HER-2 peptide vaccine, we screened a phage display 12-mer peptide library with trastuzumab as the target. A mimetic peptide (mimotope) H98 (LLGPYELWELSH) that could specifically recognize trastuzumab was isolated. The DNA encoding peptide H98 was cloned and expressed as the fusion protein GST-H98 in Escherichia coli BL21. The purified GST-H98 could specifically bind to trastuzumab and block the binding of trastuzumab to HER-2 protein. Moreover, H98 could significantly block the function of trastuzumab inhibiting the growth of cancer cells. Mice that were immunized with GST-H98 made specific antibody to H98 as well as to HER-2. In addition, T-cell proliferation occurred in mice immunized with GST-H98. Although no sequence homology was found between H98 and HER-2, through the use of structure analysis we were able to determine that peptide H98 contributed to a conformational epitope of HER-2. Furthermore, we determined that the last two amino acids at the C terminus, and the third together with the fourth amino acid at the N terminus of peptide H98 are critical to the binding of H98 to trastuzumab. As a result, we conclude that peptide H98 has potential for being developed as a HER-2 vaccine for biotherapy of cancer with HER-2 overexpression.

Animals↗
PubMed2003

Mimotopes of the Vi antigen of Salmonella enterica serovar typhi identified from phage display peptide library.

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.

Amino Acid Sequence↗
PubMed2005

SUPERFICIAL--surface mapping of proteins via structure-based peptide library design.

BACKGROUND: The determination of protein surfaces and the detection of binding sites are essential to our understanding of protein-protein interactions. Such binding sites can be characterised as linear and non-linear, the non-linear sites being prevailant. Conventional mapping techniques with arrays of synthetic peptides have limitations with regard to the location of discontinuous or non-linear binding sites of proteins. RESULTS: We present a structure-based approach to the design of peptide libraries that mimic the whole surface or a particular region of a protein. Neighbouring sequence segments are linked by short spacers to conserve local conformation. To this end, we have developed SUPERFICIAL, a program that uses protein structures as input and generates library proposals consisting of linear and non-linear peptides. This process can be influenced by a graphical user interface at different stages, from the surface computation up to the definition of spatial regions. CONCLUSION: Based on 3D structures, SUPERFICIAL may help to negotiate some of the existing limitations, since binding sites consisting of several linear pieces can now be detected.

Binding Sites↗
PubMed1991

A new type of synthetic peptide library for identifying ligand-binding activity.

Our aim was to improve techniques for drug development by facilitating the identification of small molecules that bind with high affinity to acceptor molecules (for example, cell-surface receptors, enzymes, antibodies) and so to mimic or block their interaction with the natural ligand. Previously such small molecules have been characterized individually on a serial basis. The systematic synthesis and screening of peptide libraries of defined structure represents a new approach. For relatively small libraries, predetermined sequence variations on solid-phase supports have been used, and large libraries have been produced using a bacteriophage vector into which random oligodeoxynucleotide sequences have been introduced, but these techniques have severe limitations. Here we investigate an alternative approach to synthesis and screening of peptide libraries. Our simple methodology greatly enhances the production and rapid evaluation of random libraries of millions of peptides so that acceptor-binding ligands of high affinity can be rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.

Amino Acid Sequence↗
PubMed1990

Random peptide libraries: a source of specific protein binding molecules.

Libraries of random peptide sequences were constructed and screened to identify peptides that specifically bind to proteins. In one of these about 2 X 10(7) different 15-residue peptide sequences were expressed on the surface of the coliphage M13. Each phage encoded a single random sequence and expressed it as a fusion complex with pIII, a minor coat protein present at five molecules per phage. Phage encoding nine different streptavidin-binding peptide sequences were isolated from this library. The core consensus sequence was His-Pro-Gln and binding of these phage to streptavidin was inhibited by biotin. This type of library makes it possible to identify peptides that bind to proteins (or other macromolecules) that have no previously known affinity for peptides.

Adsorption↗
PubMed2004

Identification of peptides targeting the surface of Plasmodium falciparum-infected erythrocytes using a phage display peptide library.

Drastic changes in the plasma membrane of Plasmodium falciparum-infected red blood cells (iRBCs) make the surface of iRBCs distinct from that of the uninfected erythrocyte. To identify small peptides that would specifically recognize the altered surface of iRBCs, we screened a phage display peptide library (PDL) on the surface of iRBCs. After the sixth panning of the PDL, eight phage clones of 18 sequenced clones had the same sequence, LVDAAAL (named P1) and specific binding of P1 to the surface of iRBCs was confirmed using phage expressing P1 peptides and synthetic P1 peptide. When P1 peptide was conjugated with a peptide having moderate hemolytic activity, the peptide conjugate inhibited the growth of intracellular parasites in a dose-dependent manner, whereas control peptides were without effect. Our results demonstrate that the P1 peptide may be a lead compound for the development of anti-malarial agents targeting the surface of iRBCs.

Amino Acid Sequence↗
PubMed1999

Convergent peptide libraries, or mixotopes, to elicit or to identify specific immune responses.

Many recent studies have demonstrated the flexibility of epitope recognition by the immune system. This can be explored using a particular type of combinatorial peptide library, termed as 'convergent', consisting essentially of closely related molecular species; from this a fuzzy set can be constructed, which comprises several variants of a peptide that would act in synchrony to represent a model antigen and its recognition by the immune system.

Amino Acid Sequence↗
PubMed1999

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Amino Acid Sequence↗
PubMed2000

[Epitope mapping of HIV-1 using phage-display random peptide library and the purified IgG from HIV patient].

OBJECTIVE: By using phage display random peptide library, the B cell epitope of HIV protein was studied. METHODS: The library displaying random dodecamers was biopanned first with human total IgG antibodies against HIV-1, and then non-specific phages were subtracted by purified IgG from non-HIV sera. After three rounds of screening, the positive phages were tested by ELISA for their reactivity with HIV(+)-IgG and HIV(-)-IgG antibodies. Phage that showed positive reactivity with HIV(+)-IgG, but negative to HIV(-)-IgG, were selected and their displayed peptides were determined by DNA sequencing. RESULTS: All the 13 positive clones sequenced displayed five kinds of peptides (SPKCLGKLLCAF, THQCLGKLQCGV, SCSAKFTCTTQI, KSDCSARFMCSV, DCLKQWACEWSR) that have homology to the HIV-I gp4l (602GCSGKLICTIN613). CONCLUSIONS: This method demonstrated there is a dominant epitope in the region of HIV-1 gp4l and can be used in the research of the B cell epitope of HIV protein.

Bacteriophages↗
PubMed2002

Design, synthesis and evaluation of peptide libraries as potential anti-HIV compounds, via inhibition of gp120/cell membrane interactions, using the gp120/cd4/fab17 crystal structure.

The crystal structure of a gp120/CD4/Fab17b complex was analysed leading to the design of several peptide libraries in the hope of obtaining novel gp120/cell membrane receptor interaction inhibitors, especially inhibitors of gp120/CD4 and gp120/chemokine receptor interactions. Syntheses of tri- and tetra- and pentapeptides were performed via a solid phase synthesis methodology using a Rink Amide MBHA resin and a Fmoc strategy giving C-terminal amide form peptides. Compounds were assayed against C8166 cells infected by HIV-1 IIIB and screened using a gp120 binding assay and the FIGS reporter gene assay.

Anti-HIV Agents↗
PubMed2003

Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library.

M13 phages that display random disulfide constrained heptapeptides on their gpIII proteins were used to select for high affinity ligands to hepatitis B core antigen (HBcAg). Phages bearing the amino acid sequences C-WSFFSNI-C and C-WPFWGPW-C were isolated, and a binding assay in solution showed that these phages bind tightly to full-length and truncated HBcAg with K D rel values less than 25 nM, which is at least 10 orders of magnitude higher than phage carrying the peptide sequence LLGRMK selected from a linear peptide library. Both the phages that display the constrained peptides were inhibited from binding to HBcAg particles by a monoclonal antibody that binds specifically to the immunodominant region of the particles. A synthetic heptapeptide with the amino acid sequence WSFFSNI derived from one of the fusion peptides inhibits the binding of large surface antigen (L-HBsAg) to core particles with an IC50 value of 12 +/- 2 microM. This study has identified a smaller peptide with a greater inhibitory effect on L-HBsAg-HBcAg association.

Antibodies, Monoclonal↗
PubMed2005

A random peptide library fused to CCR5 for selection of mimetopes expressed on the mammalian cell surface via retroviral vectors.

A random peptide library was expressed on the surface of a mammalian cell by applying retroviral vectors. The random sequence was fused to the CCR5 chemokine receptor, which served as a scaffold to present the library at the cell surface. We used this library to isolate an epitope mimetope in a proof of principle system. This approach can become a tool for rapid creation of peptidic expression domains in a eukaryotic environment. Applications include the creation of decoys for receptors in cell-cell interactions, screening for molecules that drive ligand expression on target cells in two-cell interaction screens, among other utilities.

Base Sequence↗
PubMed2004

Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution.

CD8 and CD4 lymphocytes control cytomegalovirus (CMV) infection in immunocompetent individuals, while patients with defective cellular immunity are prone to endogenous reactivation of latent CMV or, like seronegative subjects, prone to primary infection. Administration of CMV-specific CD8 lymphocytes was beneficial for immunocompromised hemopoietic stem cell (HSC) graft recipients. Since CD4 cells contribute to expansion of cytotoxic T lymphocytes (CTL), we defined new T(h) peptides on the immunodominant protein pp65 recognized by CD4 cells from HLA-typed subjects, in the perspective of complementing CTL administration with CMV-specific T(h) cells. Screening by ELISPOT on CD4 and CD8 subsets using overlapping peptides identified 10 novel CD4 peptides. To simplify procedures to generate T cell lines, we used a CD4 peptide library for T cell stimulation instead of ill-defined viral lysates, without the requirement of dendritic cells. This library stimulated CMV-specific CD4 cells. In fact, peptide-induced CD4 cells responded to pp65 and to the viral lysate. These cells were also devoid of alloreactivity after one stimulation cycle. Since Good Manufacturing Procedure-grade peptides can be synthesized, culture conditions are simplified and alloreactivity is rapidly lost, these procedures based on peptide stimulation can facilitate implementation of adoptive reconstitution of CD4 responses in immunocompromised patients also in the case when the HSC allodonor is available for generation of the T cell line.

Adolescent↗
PubMed2005

The use of proteotypic peptide libraries for protein identification.

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins.

Algorithms↗
PubMed2003

Mass spectrometric analysis of combinatorial peptide libraries derived from the tandem repeat unit of MUC2 mucin.

Four 19-member synthetic peptide libraries, based on the TX1TX2T epitope motif of the mucin-2 gastrointestinal glycoprotein (MUC2) and ranging in peptide length from dipeptides to 15-mers (XT, TXT, TQTXT and KVTPTPTPTGTQTXT), were synthesized by combinatorial solid phase peptide synthesis using the portioning-mixing combinatorial approach, and analysed by electrospray ionization mass spectrometry at different (1000-10000) resolutions. Most of the components of the individual libraries could be easily identified in a single-stage molecular mass screening experiment. The resolving power of the instrument becomes an important factor above 800-1000 Da molecular mass, when predominantly multiply charged molecular ions are formed. Approaches to the identification of isobars (glutamine/lysine), isomers leucine/isoleucine) and sequence variations by tandem mass spectrometry, and/or by high-performance liquid chromatography-mass spectrometry are outlined.

Amino Acid Sequence↗