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At least 307 records · Page 17Linked to original sources
PubMed2006

Discovery of galectin ligands in fully randomized combinatorial one-bead-one-compound (glyco)peptide libraries.

The involvement of human lectins (galectins) in disease progression accounts for the interest to design potent inhibitors. Three fully randomized hexa(glyco)peptide libraries were prepared using the portion mixing method combined with ladder synthesis. On-bead screening with fluorescently labelled galectin-1 and -3 yielded a series of lead structures, whose inhibitory activity on carbohydrate-dependent galectin binding was tested in solution by solid-phase and cell assays. The various data obtained define the library approach as a facile route for the discovery of selective (glyco)peptide-based galectin inhibitors.

Cell Line, Tumor↗
PubMed1992

Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage.

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.

Amino Acid Sequence↗
PubMed1997

Isolation of peptides from phage-displayed random peptide libraries that interact with the talin-binding domain of vinculin.

Peptides isolated from combinatorial libraries typically interact with, and thus help to characterize, biologically relevant binding domains of target proteins. To characterize the binding domains of the focal adhesion protein vinculin, vinculin-binding peptides were isolated from two phage-displayed random peptide libraries. Altogether, five non-similar vinculin-binding peptides were identified. Despite the lack of obvious sequence similarity between the peptides, binding and competition studies indicated that all five interact with the talin-binding domain of vinculin and do not disrupt the binding of alpha-actinin or paxillin to vinculin. The identified peptides and talin bind to vinculin in a comparable manner; both bind to immobilized vinculin, but neither binds to soluble vinculin unless the C-terminus of vinculin has been deleted. An analysis of amino acid variants of one of the peptides has revealed three non-contiguous motifs that also occur in the region of talin previously demonstrated to bind vinculin. Amino acid substitutions within a 127-residue segment of talin capable of binding vinculin confirmed the importance of two of the motifs and suggest that residues critical for binding are within a 16-residue region. This study demonstrates that the vinculin-binding peptides interact with vinculin in a biologically relevant manner and represent an excellent tool for further study of the biochemistry of vinculin.

Amino Acid Sequence↗
PubMed1999

Method for determining protein kinase substrate specificities by the phosphorylation of peptide libraries on beads, phosphate-specific staining, automated sorting, and sequencing.

A method is described for the elucidation of the peptide substrate phosphorylation specificity of a protein kinase. Peptide libraries with two to six degenerate positions and a length of seven or nine amino acids were generated directly on Sepharose beads by solid-phase peptide synthesis according to the split-and-mix procedure. The immobilized peptides were incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA) as a model enzyme resulting in the phosphorylation of the beads that contain the recognition motif of the kinase. The beads were then stained with a new phosphate-monoester-specific fluorescent dye consisting of a complex of iron(III) with fluorescein-coupled iminodiacetic acid. A flow cytometer was used to analyze the phosphorylation efficiency and the beads with the highest phosphorylation degree were isolated by the use of a fluorescence-activated cell sorter. Pool sequencing of those beads revealed the preferred kinase motif. The results are in good agreement with data from the literature. The method lends itself to the rapid elucidation of the specificity of uncharacterized protein kinases.

Amino Acid Sequence↗
PubMed1999

Characterization of cerebrospinal fluid antibody specificities in neurocysticercosis using phage display peptide library.

To identify epitopes for antibodies present in cerebrospinal fluid (CSF) samples from two patients with confirmed neurocysticercosis, we used a phage peptide library that displayed random dodecapeptides as a fusion to the minor coat protein (pIII) of phage M13. To increase the specificity of selection, plates coated with anti-human Fc antibody were used in five rounds of biopanning. The DNA inserts of 30 selected clones were determined and the deduced amino acid sequences were analyzed. Sequence similarities between affinity-selected clones and three known T. solium and T. crassiceps proteins were encountered. Two phage clones have been shown to react in enzyme-linked immunosorbent assay with CSF samples from other patients with neurocysticercosis, and no reaction was observed with CSF samples from patients with other neurological disorders used as negative controls. Our results suggest that affinity selection of peptides from phage libraries using CSF samples from patients is an attractive tool for the development of novel reagents for a simple and sensitive immunodiagnostic test and for understanding the molecular mechanisms participating in the pathogenesis of neurocysticercosis.

Amino Acid Sequence↗
PubMed2001

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Amino Acid Sequence↗
PubMed2005

[Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain].

Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.

ADAM Proteins↗
PubMed1995

Hsc70-binding peptides selected from a phage display peptide library that resemble organellar targeting sequences.

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.

Amino Acid Sequence↗
PubMed2000

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9 / 1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Amino Acid Sequence↗
PubMed2005

Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries.

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.

Bacterial Proteins↗
PubMed2000

The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach.

The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.

Amino Acids↗
PubMed2004

Biodistribution of filamentous phage peptide libraries in mice.

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens. Phage selections in animals have been performed for very short times without optimization for biodistribution or clearance rates to a particular organ. It is hypothesized that peptides that home to a desired antigen/organ can be obtained from in vivo phage experiments by optimization of incubation times, phage extraction and propagation procedures. To accomplish this goal, one must first gain a better understanding of the in vivo biodistribution and rate of clearance of engineered phage peptide display libraries. While the fate of wild type phage in rodents has been reported, the in vivo biodistribution of the commonly used engineered fd-tet M13 phage peptide display libraries (such as in the fUSE5 vector system) have not been well established. Here we report the biodistribution and clearance properties of fd-tet fifteen amino acid random peptide display libraries in fUSE5 phage in three common mouse models employed for drug discovery - CF-1, nude, and SCID mice.

Animals↗
PubMed2006

Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein.

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.

Amino Acid Sequence↗
PubMed1994

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Amino Acid Sequence↗
PubMed1999

Vascular targeting with phage peptide libraries.

We have developed an in vivo selection system in which phage capable of selective homing to different tissues are recovered from a phage display peptide library following intravenous administration. Using this strategy, we have isolated several organ and tumor-homing peptides. We have shown that each of those peptides binds to different receptors that are selectively expressed on the vasculature of the target tissue. The tumor-homing peptides bind to receptors that are upregulated in tumor angiogenic vasculature. Targeted delivery of doxorubicin to angiogenic vasculature using these peptides in animal models decreased toxicity and increased the therapeutic efficacy of the drug. Vascular targeting may facilitate the development of other treatment strategies that rely on inhibition of angiogenesis and lead to advances in cancer treatment. Our technology is also likely to extend the potential for targeting of drugs, genes, and radionuclides in the context of many diseases.

Animals↗
PubMed2001

Determination of protease cleavage site motifs using mixture-based oriented peptide libraries.

The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates.

Amino Acid Motifs↗
PubMed1994

Constrained peptide libraries as a tool for finding mimotopes.

The monoclonal antibody (mAb) KAA8 recognizes the peptide angiotensin II (AII). The KAA8 mAb was biopanned using two phage-displayed peptide libraries, one unconstrained, the other constrained by a disulfide bond. After several cycles of biopanning, both libraries showed enrichment for phage that bind KAA8. Phage isolated from the unconstrained library contain a consensus sequence that matches the sequence of AII. A consensus sequence was also identified from the constrained library that does not resemble the AII sequence, and represents a mimotope of AII. We have also demonstrated that monovalent phage display can be used to discriminate between modest and high-affinity binding peptides.

Amino Acid Sequence↗
PubMed1997

Erythromycin resistance peptides selected from random peptide libraries.

Translation of a 5-codon mini-gene encoded in Escherichia coli 23 S rRNA was previously shown to render cells resistant to erythromycin (Tenson, T., DeBlasio, A., and Mankin, A. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5641-5646). Erythromycin resistance was mediated by a specific interaction of the 23 S rRNA-encoded pentapeptide with the ribosome. In the present study, peptides conferring erythromycin resistance were selected from in vivo expressed random peptide libraries to study structural features important for peptide activity. Screening of a 21-codon mini-gene library (the general structure ATG (NNN)20 TAA) demonstrated that only short peptides (3-6 amino acids long) conferred erythromycin resistance. Sequence comparison of erythromycin resistance peptides isolated from the 5-codon library (ATG (NNN)4 TAA) revealed a strong preference for leucine or isoleucine as a third amino acid and a hydrophobic amino acid at the C terminus of the peptide. When tested against other antibiotics, erythromycin resistance peptides rendered cells resistant to other macrolides, oleandomycin and spiramycin, but not to chloramphenicol or clindamycin. Defining the consensus amino acid sequence of erythromycin resistance peptides provided insights into a possible mode of peptide action and the nature of the peptide binding site on the ribosome.

Chromosome Mapping↗