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PubMed2001

Construction of high-complexity combinatorial phage display peptide libraries.

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.

Amino Acid Sequence↗
PubMed2002

Construction of the novel conformationally-restricted peptide library for screening of peptides that control the interaction between nucleobases.

A unique conformationally-restricted peptide library was constructed using a loop structure as a structural scaffold. This library was used for the screening of the amino acid sequences that control the interaction between nucleobase triplets. The peptides have PNAs at the C-terminus as the recognition site and the random amino acid sequence at the N-terminus as the effector for the interaction between PNA and its complementary DNA triplets. From the peptide libraries constructed by the positional scanning method, the sequences that affect the interaction between PNA and complementary DNA were selected. The difference in the characteristic results by using A-T and G-C pairs was presented. This study would also give us some useful information about interaction between peptides and nucleic acids, such as relevances between these biomolecules in a prebiotic era.

Animals↗
PubMed2002

Immunogenically fit subunit vaccine components via epitope discovery from natural peptide libraries.

Antigenic peptides that bind pathogen-specific Abs are a potential source of subunit vaccine components. To be effective the peptides must be immunogenically fit: when used as immunogens they must elicit Abs that cross-react with native intact pathogen. In this study, antigenic peptides obtained from phage display libraries through epitope discovery were systematically examined for immunogenic fitness. Peptides selected from random peptide libraries, in which the phage-displayed peptides are encoded by synthetic degenerate oligonucleotides, had marginal immunogenic fitness. In contrast, 50% of the peptides selected from a natural peptide library, in which phage display segments of actual pathogen polypeptides, proved very successful. Epitope discovery from natural peptide libraries is a promising route to subunit vaccines.

Amino Acid Sequence↗
PubMed1994

Major histocompatibility complex class I allele-specific peptide libraries: identification of peptides that mimic an H-Y T cell epitope.

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.

Amino Acid Sequence↗
PubMed1999

The basic structure of filamentous phage and its use in the display of combinatorial peptide libraries.

Combinatorial peptide libraries have been playing a major role in the search for new drugs, ligands, enzyme substrates, and other specifically interacting molecules. The principal features of these libraries require a versatile repertoire, an easily identifiable tag for each of the library members, a simple method of synthesis, and a compatibility with the biochemical milieu. Two types of combinatorial libraries are in use: synthetic libraries and biological (mainly phage display) ones. An advantage of the biological libraries is due to the ability of each of the library members to replicate itself and to the fact that they carry their own coding sequences. The uniqueness of filamentous phage is that of its five virion proteins, three can tolerate the insertion of foreign peptides, each in a distinctive manner. The major coat protein, pVIII, is capable of displaying hundreds of peptide copies over the phage virion, pIII can display either one or five copies, and pVI, as opposed to the first two, displays its peptides such that the carboxy terminus is oriented outward. A major drawback of filamentous phage is its size. The length of an intact phage particle is 930 nm and it contains an ssDNA of 6400 bp. 2800 copies of the major coat protein form a "fish scale" cover over most of the virion DNA, whereas five copies of pIII, which has been the major protein used for library display, and five copies of pVI are located at one end of the filamentous virion. There is no doubt that in order to improve the quality of filamentous phage libraries, the size of phage should be drastically reduced. Comprehensive research on the phage life cycle and its structure will lead us to the construction of miniature phage and to other methods that will enable an in vivo expanding of the library repertoire as well as to binding-induced specific clone-proliferation.

Bacteriophages↗
PubMed1996

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Animals↗
PubMed1994

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Amino Acid Sequence↗
PubMed1995

Use of a constrain phage displayed-peptide library for the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7).

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.

Amino Acid Sequence↗
PubMed2005

A convenient method for synthesis of cyclic peptide libraries.

Cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. Owing to the robustness of amide bond chemistry, the ability to explore extensive chemical diversity by incorporation of unnatural and natural amino acids, and the ability to explore conformational diversity, through the incorporation of various constraints, arrays of cyclic peptides can be tailored to broadly sample chemical diversity. We describe the combination of a safety catch linker with a directed-sorted procedure for the synthesis of large arrays of diverse cyclic peptides for high-throughput screening.

Amino Acids↗
PubMedDate not supplied

[Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library].

Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.

Amino Acid Sequence↗
PubMedDate not supplied

Delineation of selective cyclic GMP-dependent protein kinase Ialpha substrate and inhibitor peptides based on combinatorial peptide libraries on paper.

Peptide libraries on cellulose paper have proven to be valuable tools for the a priori determination of substrate specificities of cyclic AMP- and cyclic GMP-dependent protein kinases (cAMP-kinase and cGMP-kinase) on the basis of octa-peptide sequences. Here, we report the extension of our peptide library screens to 12-mer and 14-mer peptide sequences, resulting in highly cGMP-kinase Ialpha selective peptides. The sequences TQAKRKKSLAMA-amide and TQAKRKKSLAMFLR-amide, with Km values for cGMP-kinase Ialpha of 0.7 and 0.26 microM and Vmax values of 11.5 and 10.9 micromol/min/mg, respectively, display a high specificity for this enzyme. Furthermore, replacing the phosphate acceptor residue serine with alanine in TQAKRKKSLAMA-amide resulted in the highly cGMP-kinase Ialpha selective inhibitor peptide TQAKRKKALAMA-amide, with inhibitor constants for cGMP-kinase Ialpha and cAMP-kinase of 7.5 microM and 750 microM, respectively. Selective cGMP-kinase inhibitors have the potential to play an important role in the elucidation of the distinct cellular functions of cGMP-kinase separate from those activated by cAMP-kinases, and, therefore, may play an important role as pharmaceutical targets. Molecular docking experiments of the most cGMP-kinase selective sequences on a molecular model of the catalytic domain of cGMP-kinase Ialpha suggest that they adopt unique conformations, which differ significantly from those observed for the cAMP-kinase-specific inhibitor PKI(5-24). Our results suggest that despite their structural similarities, cAMP-kinase and cGMP-kinase use distinct peptide substrate and inhibitor conformations, which could account for their unique substrate specificities. These findings are further supported by cAMP- and cGMP-kinase-selective inhibitor analogs with (D)-Ala residues at the inhibitory positions.

Animals↗
PubMed1999

Affinity chromatographic screening of soluble combinatorial peptide libraries.

Affinity chromatography using immobilized S-protein was used for the screening of affinity peptide ligands from two soluble peptide libraries. Peptide library I consisted of octamers with glycine (G) at both termini of each peptide, i.e. GXXXXXXG. The six center positions were constructed using random sequences of six L-amino acids (Y, N, F, E, V, and L). Peptide library II also consisted of octamers but with glycine and valine (V) at both termini of each peptide (GVZZZZVG). The four variable center positions of peptide library II were random sequences of 18 L-amino acids. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of peptide libraries I and II resulted in 12 and 7 affinity peptides, respectively. Eight out of the twelve peptides from peptide library I contained the clear consensus sequence NFEV. Peptide library II resulted in affinity peptides with the sequences GVNFEVVG, GVNFTVVG and GVFFEL(I)VG. The advantages and limitations of affinity chromatography in peptide library screening are discussed.

Amino Acid Sequence↗
PubMed1996

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries.

Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10(7) hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.

Amino Acid Sequence↗
PubMed1995

Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.

Amino Acid Sequence↗
PubMedDate not supplied

Docking of combinatorial peptide libraries into a broadly cross-reactive human IgM.

A monoclonal IgM cryoglobulin with diverse binding behavior was isolated from a patient (Mez) with Waldenström's macroglobulinemia. It gave very high titers in the binding of combinatorially synthesized libraries of peptides ranging in size from two to eight residues. The crystal structure of Mez Fv revealed that the binding site was divided into two cavities of unequal volumes with dimensions and chemical properties that were compatible with the binding of peptides. Access to this unique combination of structural information and peptide binding data led us to carry out Mez-peptide docking simulations to gain insight into the Mez binding propensities. In the present article, the results for docking of five peptide libraries are combined with discussions of the methods and approximations involved in the docking process. We analyze the origins of peptide binding affinity for Mez IgM in terms of its cross-reactivity and its structural preferences.

Antibodies, Monoclonal↗
PubMed1998

A peptidomimetic antagonist of TNF-alpha-mediated cytotoxicity identified from a phage-displayed random peptide library.

Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-alpha antagonists. After direct interaction of recombinant human TNF-alpha with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-alpha-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-alpha using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-alpha-induced cytotoxicity in vitro in a dose-dependent fashion. These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-alpha.

Amino Acid Sequence↗
PubMed1995

Novel avidin and streptavidin binding sequences found in synthetic peptide libraries.

Synthetic resin-bound peptide libraries made of protein L-amino acids have been synthesized. During screening of the libraries, peptides that bind to avidin have been identified containing a novel motif with two histidines separated by one residue. A sub-library was synthesized and screened, and new critical residues appeared surrounding the two histidines. Additionally peptide libraries made of D-amino acids have been screened with avidin and streptavidin and novel motifs have been found.

Amino Acid Sequence↗
PubMed2000

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

Animals↗