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PubMedDate not supplied

Development of tissue plasminogen activator specific "on demand cleavable" (odc) linkers for radioimmunotherapy by screening one-bead-one-compound combinatorial peptide libraries.

New strategies are needed to protect normal organs from radiation in cancer radioimmunotherapy (RIT). This can be achieved by rapid clearance of radiometal in the circulation after accumulation of radioimmunoconjugates (RIC) in the tumor. Our strategy is to place highly efficient and specific cleavable linkers between radiometal chelates and the tumor targeting agents. Such linkers must be resistant to cleavage by enzymes present in the plasma and the tumor. After radiotargeting agents have accumulated in the tumor, a cleaving agent can be administered "on demand" to cleave a specific linker, resulting in the release of radiometal from the circulating RIC in a form that will have rapid renal clearance. We have selected TNKase, a thrombolytic agent approved for patient use, as our model on-demand cleaving agent. To identify TNKase-specific linkers, we screened fluorescent-quenched random "one-bead-one-compound" (OBOC) combinatorial peptide libraries. d-Amino acid containing peptides that were specific for TNKase but were resistant to cleavage by plasma and tumor-associated proteases were identified. One of these peptide substrates (rqYKYkf) was used to link the DOTA chelate to ChL6, a monoclonal antibody known to target breast cancer. This antibody conjugate was stable in plasma for 7 days while preserving the immunoreactivity to intact tumor cells. The addition of TNKase at clinical achievable plasma level (10 mug/mL) resulted in the release of 28% of the radiometal from the radioimmunoconjugate within 72 h. This lead linker, after further optimization to increase its response to TNKase, may be useful in the development of more effective radioimmunotherapeutic and radioimaging agents.

Cellulose↗
PubMed1998

Identification of epitopes in cucumber mosaic virus using a phage-displayed random peptide library.

Antigenic sites in the cucumber mosaic virus (CMV) coat protein (CP) have been identified using a polyclonal antiserum prepared against glutaraldehyde-fixed virions. Antibodies were used to screen a random peptide library of heptamers displayed on the surface of a bacteriophage. Eight of 36 (22%) sequenced phage clones had inserts resembling a putative virion surface domain of the CMV CP. This region has the sequence LETDEL, corresponding to amino acids 194-199 in the Fny-CMV CP. The binding of phage clones to Fny-CMV antiserum was inhibited by a synthetic peptide representing this region. Six of 36 (17%) phage clones contained sequences corresponding to a C-terminal sequence in the Fny-CMV CP, which is thought to be internal in assembled virions. This sequence, EHQRIPTSGV, represents amino acids 206-215 and all but the P residue were observed in at least one clone. Four of 36 (11%) sequenced phage clones carried sequences that matched a portion of the sequence RLLLPDSV, corresponding to amino acids 89-96 in the Fny-CMV CP. This region was also identified as the antigenic site recognized by a monoclonal antibody (MAb23C10E4). Eleven percent of the phage (4 of 36) contained sequences matching at least three amino acids of the N-terminal region in the CMV CP. The positions of the antigenic sites seen in this study are consistent with a predicted structure for the CMV CP.

Antibodies, Monoclonal↗
PubMed2002

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

Amino Acid Motifs↗
PubMed2003

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

Amino Acid Sequence↗
PubMed2004

A subtractive fluorescence-activated cell-sorting strategy to identify mimotopes of HBV-preS protein from bacterially displayed peptide library.

A novel subtractive fluorescence-activated cell-sorting (FACS) strategy using a model system is described here to identify disease-specific (DS) epitopes from a bacterially displayed random peptide library. In this process, preimmune serum was used as "Driver " to block any common binding sites on the bacterial surface and the labeled anti-preS IgG polyclonal antibodies from immunized serum were used as "Tester" to enrich preS-specific mimotopes. Bacterial clones were identified out of this pool through an "antigen-independent" procedure only using both different sera samples. After four rounds of sub-FACS screening, 41 out of 50 bacterial clones were identified as reacting with the immunized serum but not reacting with the pre-immune one. Two motif sequences HQLD and DPAF were obtained from 13 clones. Immunization of mice with two representative bacterial clones elicited a strong specific response against native preS antigen in comparison with the control. This technique may provide a useful technology platform for high-throughput screening of disease-related epitope which is of importance to develop vaccine against some infectious diseases whose pathogen or immunodominant antigen is still unknown.

Animals↗
PubMed1998

Screening of Cellulose Binding Motif (CBM) from Phage Peptides Library.

Bacteriophages capable of binding cellulose matrix were screened from a 15-mer phage peptides library. In the deduced amino acid sequences of the screened phages, a characteristic region containing a conserved aromatic residue [tyrosine (Y) or phenylalanine (F)] was found, which is similar to the normal cellulose binding domains found in fungal and bacterial cellulose catalysase. Dot-ELISA showed that the phage containing sequence as SWYL has higher affinity to cellulose fibre than other phages, such as those containing sequences as CWYGNC, CWYGEC and XSWYDXXSWFSX. Results indicated that SWYL maybe a good candidate for cellulose binding motif. This work laid basis for further study on cellulose binding motif.

Journal Article↗
PubMed1990

Fragment peptide library for classification and functional prediction of proteins.

From protein sequence comparison data found in the literature, a library was organized using peptide fragment sequences which are common to related proteins. Each of the fragments was then examined for its occurrence in all the protein superfamilies defined by the NBRF-PIR data base. We have selected those fragment peptides that appear exclusively in one or a few superfamilies, and thus made a library of fragment peptides that characterize specific superfamilies. Such characteristic peptides are, in general, five to seven residues long and contain unusually high proportions of glycine and cysteine. This collection is a useful resource for the classification and functional prediction of protein molecules.

Amino Acid Sequence↗
PubMed2006

[Identification of mimotope peptides which bind to the SARS-CoV spike protein specific monoclonal antibody 2C5 with phage-displayed peptides library].

To identify the epitope of SARS-CoV spike protein specific neutralizing monoclonal antibody (MAb) 2C5. The antibody was used as target and three rounds of bio-panning were conducted with phage-display peptide library. After the third panning, 20 phage-plague clones were randomly picked and analyzed for the binding ability with the MAb 2C5 by ELISA. The display sequence analysis demonstrated that among the twenty phage clones, eight clones displayed the same seven-peptide TPEQQFT. All these eight phage-clones showed strongest binding activity with 2C5 in phage ELISA analysis. Furthermore, phages displaying peptide TPEQQFT could specifically inhibit the binding of MAb 2C5 with SARS-CoV spike protein. The results demonstrated that TPEQQFT is a mimic epitope peptide containing neutralizing MAb 2C5. This study may provide information for further structural and functional analysis of spike protein and development vaccine for severe acute respiratory syndrome.

Amino Acid Sequence↗
PubMed2002

Optimisation of a peptide library for screening specific RNA ligands by flow-injection NMR.

Flow-injection NMR is a very attractive technique for identifying weak but specific ligand-macromolecule interactions. The low-sensitivity of this method is, however, a major concern which hinders its widespread application to large-scale screening. Focussing on RNA-targeting ligands, we have shown in this report that it is possible to design a peptide library that will simultaneously satisfy a number of physico-chemical restraints and sample the sequence space in an "optimal" fashion. This library, which contains less than 200 peptides, covers most of the allowable tripeptide sequences in a non-redundant fashion. In addition, for almost any allowable tetrapeptide sequences, the library contains a close neighbor that will differ at the most by a single conservative replacement. A subset of this library was actually synthesised and used in a preliminary screen against human tRNA(Lys)(3), the primer of HIV-1 reverse transcriptase.

Algorithms↗
PubMedDate not supplied

Characterization of the epitope for 4C4.1 mAb on alpha-latrotoxin using phage display-peptide libraries: prevention of toxin-dependent 45Ca(2+) uptake in non-neuronal human embryonic cells transiently expressing latrophilin.

alpha-Latrotoxin, a protein toxin present in the venom of black widow spider, interacts with membrane receptors of neurons and other secretory cells to stimulate exocytosis. Two types of receptors have been identified and cloned. Our attention has been focused on the calcium independent receptor, a G-protein coupled receptor, named latrophilin to see whether alpha-latrotoxin interaction was capable to produce an ionotropic effect, in alternative to the metabotropic hypothesis. Expression of latrophilin receptor is sufficient for the alpha-latrotoxin effect to become manifest. By inducing the transient expression of latrophilin receptor in non-neuronal human embryonic cells, we made them susceptible to toxin action as demonstrated by the increase in 45Ca(2+) accumulation detected after toxin treatment. Since the presence of a monoclonal antibody against alpha-latrotoxin (4C4.1 mAb) was able to obliterate toxin-dependent effects, we further investigated the nature of toxin-antibody interaction by characterization of the binding epitope using phage display-peptide libraries. A conformational epitope was recognized and partially localized on a region of the peptide toxin whereby a tetrameric structure is formed and inserted into the membrane of target cells where it functions as a pore.

Animals↗
PubMed1999

Use of a 49-peptide library for a qualitative and quantitative determination of pseudomonal serralysin specificity.

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z-AXXA (amide) (X = A, L, V, F, S, R, E). The library was analyzed by LC-MS before and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X-X peptide bond, even if in some cases, A-X and X-A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P1' position. The results obtained suggest that the specificity in the P1' position, where an hydrophobic residue was preferentially found, seems more selective that in the Pn position. The P1 position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P1' and P1 residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor.

Amino Acid Sequence↗
PubMed1993

Biotin binders selected from a random peptide library expressed on phage.

Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed on the surface of filamentous phage. Phage binding to biotinylated proteins was half-maximally inhibited by micromolar concentrations of a monobiotinylated molecule. Sequencing of the peptide inserts of selected phages led to the identification of a previously unknown biotin-binding motif, CXWXPPF(K or R)XXC. A synthetic peptide containing this sequence motif inhibited streptavidin binding to biotinylated BSA with an IC50 of 50 microM. This compound represents the shortest non-avidin biotin-binding peptide identified to date. Our results illustrate that phage display technology can be used to identify novel ligands for a small non-proteinaceous molecule.

Amino Acid Sequence↗
PubMed1995

Isolation of antigenic mimics of MDR1-P-glycoprotein by phage-displayed peptide libraries.

To identify an MC57 epitope which is more efficiently expressed on inactivated forms of P-glycoprotein we utilized peptide libraries displayed on filamentous phage. Using this technology, we selected specific phage clones blocking the binding of the murine monoclonal (MAb) MC57 with live human multi-drug-resistant (MDR) cells, and sequenced and analyzed their DNA. The results we obtained indicate that MAb MC57 epitope could be formed by 2 regions localized on the predicted fourth and sixth extracellular loops of the current 12-transmembrane-domain model predicted for MDR1-P-glycoprotein. Surprisingly, a third region, defined by residues 800-807 of the MDR1-P-glycoprotein sequence and postulated to be intracellular, was also identified as a putative part of the MC57 epitope. This finding adds weight to the interesting hypothesis that a P-glycoprotein structure different from the current model may exist.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Affinity purification of von Willebrand factor using ligands derived from peptide libraries.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

Adsorption↗
PubMed2005

Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form.

Animals↗
PubMed2000

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries.

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Amino Acid Sequence↗
PubMed1994

The characterisation of p53 binding phage isolated from phage peptide display libraries.

Peptide phage display libraries were screened for peptides that bind to the tumour suppressor protein, human p53. Three p53 binding peptides were isolated respectively from hexamer (6-mer), dodecamer (12-mer) and icosomer (20-mer) libraries. We have characterised their interaction with p53 in detail. The phage appear to bind regions on native p53 common between mouse and man. Two conformation-specific anti-p53 monoclonal antibodies were used to dissect the phage-p53 interaction, the phage were found to preferentially bind the PAb1620-p53 conformation rather than the PAb240-p53 conformation. Mapping experiments indicated the C-terminal 30 amino acid residues of p53 were dispensable for phage binding and that the binding of SV40 large T-antigen and the phage were not mutually exclusive. Interestingly the phage were seen to exhibit differential binding to wild-type human p53 over the two point mutant p53 proteins, His175 and Trp248. Ultimately the phage appear to selectively target native wild-type p53, mimicking the specificity of SV40 large T-antigen. The ability to target specific sub-populations of p53 could be an important step in the development of therapeutics for the treatment of p53-based human malignancy.

Amino Acid Sequence↗