Biotin binders selected from a random peptide library expressed on phage.
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Amino Acid Sequence↗
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Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.
One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.
Animals↗
Potential GM-CSF Antagonists Selected from a Peptide Phage Display Library.
Peptide phage display libraries have been successfully applied in areas of mapping antibody epitope, finding ligands for enzymes, receptors, and many other molecules. But it has been demonstrated to be very difficult to select cytokine-binders from peptide phage display libraries probably because cytokine is not so sticky as antibody that there are rare chances of capturing peptide phages during biopanning. A pVIII-based peptide phage display library was panned with the cytokine GM-CSF and some GM-CSF binding clones were selected based on high throughput screening (HTS) method and confirmed by ELISA and micropanning assays. These cytokine-binders may be utilized in affinity chromatography in cytokine downstream processing and even act as potential antagonists of GM-CSF if their affinity are further improved through secondary library strategy.
Journal Article↗
Antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of Entamoeba histolytica.
Entamoeba histolytica is the protozoan parasite responsible for intestinal amoebiasis and amoebic liver abscess, which cause significant morbidity and mortality in many countries of the world. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) represent dominant surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against these components protected SCID mice from amoebic liver abscess, so PPGs might be regarded as vaccine candidates; however, their structure is very complex and only known in part. They are glycosylphosphatidylinositol-linked polypeptides of unknown sequence carrying glycan side-chains linked to serine residues via phosphodiester bonds. In order to identify peptide mimics of the E. histolytica PPG antigens, we screened six different phage-displayed random peptide libraries with the antibody EH5. Various peptide mimics of different length were identified and, in all the peptides, a distinct consensus sequence Gly-Thr-His-Pro-X-Leu could be identified. The phages strongly bound to the antibody, and the natural antigen inhibited binding of the phages to antibody EH5. In addition, several of the phages induced a significant immunoglobulin G response against amoebic antigens in immunized mice.
Amino Acid Motifs↗
Artificial aldolases from peptide dendrimer combinatorial libraries.
Peptide dendrimers were investigated as synthetic models for aldolase enzymes. Combinatorial libraries were prepared with aldolase active residues such as lysine and proline placed at the dendrimer core or near the surface. On-bead selection for aldolase activity was carried out using the dye-labelled 1,3-diketone 1a, suitable for covalent trapping of enamine-reactive side-chains, and the fluorogenic enolization probe 6. Aldolase dendrimers catalyzed the aldol reaction of acetone, dihydroxyacetone and cyclohexanone with nitrobenzaldehyde. Much like enzymes, the dendrimers exhibited strong aldolase activity in aqueous medium, but were also active in organic solvent. Dendrimer-catalyzed aldol reactions reached complete conversion in 3 h at 25 degrees C with 1 mol% catalyst and gave aldol products with up to 65% ee. A positive dendritic effect in catalysis was observed with both lysine and proline based aldolase dendrimer catalysts.
Amino Acids↗
New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries.
Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cell's surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.
Animals↗
[Application of peptide phage libraries for obtaining peptides specific to mouse lung adenocarcinoma].
Peptides binding, in vivo, with mouse lung adenocarcinoma, were selected from a peptide phage library containing above 100 million of different permutations. The selected phages carrying specific peptides accumulated in the tumor node, after intravenous injections made in A/Sn mice with induced adenocarcinoma, and persisted there even in 24 h after injections; whereas, they were detected in small quantities or not detected at all in other tissues (e.g. lungs and muscles). The selected bacteriophages were shown to accumulate not only in the primary tumor node but also in the lung with multiple metastases. Finally, amino acid sequences of exposed peptides were defined.
Adenocarcinoma↗
Pharmacologically distinct binding sites in rat brain for [3H]thyrotropin-releasing hormone (TRH) and [3H][3-methyl-histidine(2)]TRH.
We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [3H][3-Me-His(2)]TRH or [3H]TRH binding to rat cortical homogenates. [3H][3-Me-His(2)]TRH was observed to bind to a single population of high-affinity, low-capacity sites (K(d): 4.54+/-0.62 nM, N=5; B(max): 4.38+/-0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K(i) of 22 nM. K(i) values for the library peptides at [3H][3-Me-His(2)]TRH-labelled sites varied from 10(-3) to 10(-9)M; the potency order was: [3-Me-His(2)]>His>Thi>Leu,Phe,Asn>Gln, Arg, Thr, Ala, HomoPhe. All other replacements had K(i) values >10(-4)M. [3H]TRH was observed to label a single population of low-affinity, high-capacity sites (K(d): 7.55+/-1.23 microM, N=6; B(max): 3.40+/-0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [3H]TRH-labelled sites did not correlate with their affinities for [3H][3-Me-His(2)]TRH-labelled sites (r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme (r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [3H]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.
Animals↗
Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach.
BACKGROUND: Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E. RESULTS: An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies. CONCLUSIONS: Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.
Amino Acid Motifs↗
Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.
A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.
Antigens, Neoplasm↗
Experimental models of protein-RNA interaction: isolation and analyses of tRNA(Phe) and U1 snRNA-binding peptides from bacteriophage display libraries.
Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA(Phe) (tRNA(ACPhe)) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd approximately 0.1-5.0 microM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein-RNA complexes.
Amino Acid Sequence↗
Profiling the immune responses in patient sera with peptide and cDNA display libraries.
Peptide and cDNA phage display libraries can be used to determine the specificities of antibodies present in the whole sera of patients where information about the parental antigens is unknown. In this respect, patient serum antibody binding ligands have been identified. Such ligands would facilitate the design of diagnostic assays and therapeutic vaccines. In the case of cancer, this novel technology is expected to improve our understanding of the immune responses against tumour cells and to discriminate between autoantigen and true tumour specific antigens. Here, we review work on how peptide and cDNA phage display libraries can be used to address the specificity of the immune responses in patients with autoimmune diseases and cancer.
Animals↗
Identification of substrate-analog trypsin inhibitors through the screening of synthetic peptide combinatorial libraries.
Synthetic peptide combinatorial libraries (SPCLs), which are made up in total of tens to hundreds of millions of peptides, enable the systematic screening for biologically active peptides in virtually all in vitro and even in vivo assay systems. In the current study, the applicability of this method to the identification of peptide enzyme inhibitors was investigated using trypsin as the model enzyme. A specifically designed library of hexapeptide mixtures was synthesized on cotton carriers and screened. The synthetic approach, using cotton as a solid support, was modified so that the deprotected peptides remained attached to the cotton carrier until they were released into solution directly prior to being assayed. Following an iterative process of synthesis and screening, in which all of the positions of the sequence were successively defined, a number of individual hexapeptides with trypsin inhibitory activity were identified. The most active, defined individual peptide sequence was then reincorporated into a new library, now made up of dodecapeptide mixtures. The iterative screening and synthesis of this library led to a dodecapeptide with improved inhibitory activity when compared to the hexapeptide from which it was derived.
Amino Acid Sequence↗
Identification and characterization of a peptide mimetic that may detect a species of disease-associated anticardiolipin antibodies in patients with the antiphospholipid syndrome.
OBJECTIVE: To test the feasibility of applying a mimetic (specific for a patient-derived prothrombotic anticardiolipin antibody [aCL]) to study the homologous, disease-associated aCL in patients with antiphospholipid syndrome (APS). METHODS: We used the CL15 monoclonal aCL to screen 17 phage-display peptide libraries. Peptides (corresponding to recurrent peptide sequences) and their derivatives were synthesized and analyzed for binding to CL15 and for their abilities to inhibit CL15 from binding to cardiolipin. A peptide was chosen and used to study CL15-like IgG aCL in plasma samples from patients with APS, patients with systemic lupus erythematosus (SLE) but without APS, and normal healthy donors. RESULTS: Library screening with CL15 yielded 4 recurrent peptide sequences. Analyses of peptides showed that peptide CL154C reacted with antibody CL15 and inhibited binding of CL15 to cardiolipin, indicating that peptide CL154C may be a peptide mimetic for the CL15 aCL. Initial studies with plasma samples revealed that CL154C-reactive IgG was present (positivity defined as the mean + 3 SD optical density of the 25 normal controls) in 15 of 21 APS patients and 1 of 12 SLE patients. CONCLUSION: These findings suggest that it is feasible to develop a specific enzyme-linked immunosorbent assay for each immunologically and functionally distinct disease-associated aCL. Additional testing of CL154C with a larger number of APS patients and SLE patients, as well as identification of peptide mimetics for each distinct aCL, will reveal the diagnostic potential of CL154C and other mimetics in identifying patients with aCL who are at risk of developing life-threatening thrombosis.
Adolescent↗
Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries.
The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 micromol ml(-1), showed a maximum capacity of 9.1 mg Mab ml(-1) and a dynamic capacity of 3.9 mg Mab ml(-1). A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.
Antibodies, Monoclonal↗
A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.
We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.
Amino Acid Sequence↗
Peptides binding to a Gb3 mimic selected from a phage library.
Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.
Amino Acid Sequence↗
Recent advances in drug design methods: where will they lead?
Drug design methods have made significant new advances over the last ten years, mainly in the areas of molecular modelling. In more recent times important developments in theory have led to a different type of modelling becoming possible, the so-called de novo or automated design algorithms. In this new method the programs perform much of the chemist's thinking, in finding appropriately sized chemical groups to fit into a target site. However this is a combinatoric problem which has no general analytical solution; it is ripe for optimization. Other advances, such as combinatorial chemical synthesis and screening, will dramatically influence the search for new lead structures for target sites, which at present are poorly understood. Already these methods are being applied to peptide libraries. Peptides do not make good drug compounds because of their poor bioavailability; further, their flexibility reduces their affinity. In some cases peptide backbones can be removed and replaced with rigid non-peptide scaffolds.
Drug Design↗