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At least 469 records · Page 26Linked to original sources
PubMed1997

Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a versatile tool for the purification of proteins with affinity for immobilized metals. Moreover, this technique has also been used for the separation of proteins that do not exhibit significant metal affinity in the native form, by their fusion to a short metal-binding peptide (a tail), most commonly, a sequence consisting of six adjacent histidine residues (His6). A phage-displayed random hexamer library is used to select for peptides with affinity for immobilized copper. The study follows our previous investigation in which a stringent selection protocol led to the selection of only one copper-binding peptide containing two histidines. The less stringent conditions employed in this work resulted in the selection of a more diverse population of peptides, but again, dominated by peptides containing two histidines (13 out of 19). The prevalence of peptides with two histidines, in contrast to peptides with a higher number of histidines (e.g. His6 or HHHMVH), is explained based on the differences in the pH dependence of their affinity for copper. As discussed, the selected peptides with two histidines will be superior affinity tails than peptides with a higher histidine content (e.g. His6). Moreover, a peptide with a single histidine but with a very high copper affinity, is also identified. Its high copper affinity is related to the presence of several hydrophobic residues in the neighborhood of histidine. Chromatography of human interleukin-1 beta (hIL-1 beta) and several other proteins containing a single surface-exposed histidine surrounded by several hydrophobic residues confirmed that such a sequence could also serve as a very effective metal binding domain for protein purification using immobilized copper(II) columns.

Affinity Labels↗
PubMed2006

Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians.

A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.

Amino Acid Sequence↗
PubMed1996

Reproducing the immune response against the Plasmodium vivax merozoite surface protein 1 with mimotopes selected from a phage-displayed peptide library.

We have used phage display technology to identify peptides binding D14-3, a monoclonal antibody raised against the M(r) 42,000 C-terminal fragment of Plasmodium vivax merozoite surface protein 1 (PvMSP1). By screening a constrained hexapeptide library, seven independent clones binding D14-3 were isolated. The reactivity of D14-3 for these peptides was lower than for the natural antigen and the antibody binding was strictly associated with the viral context and the peptide conformation. Sequence analysis showed that five of them shared homology with the M(r) 42,000 C-terminal fragment (Pv42) and therefore appears to identify the D14-3 epitope. However, the other two peptides, while related to each other, did not correspond to any sequence in the Pv42 molecules. To evaluate their immunological interest, these phagotopes were injected into mice belonging to Balb/c, IC57BI/6 and Biozzi strains. All animals developed a strong immune response against phage particles but only Biozzi mice produced antibodies cross-reacting with Pv42. All phagotopes in Biozzi mice elicited a specific response against Pv42, even those sharing no sequence similarity with the antigen. Moreover, the avidities of these immune sera and the polyclonal response against Pv42 were comparable, suggesting phagotopes could be used as components of a subunit vaccine based on the C-terminal fragment of MSP1.

Amino Acid Sequence↗
PubMed2005

Analyte peptidomimetics selected from phage display peptide libraries: a systematic strategy for the development of environmental immunoassays.

Due to their simplicity, speed, low cost, and specificity, immunoassays have become a useful tool for the analysis of environmental pollutants. Once the anti-hapten antibodies are produced, the same hapten or a related molecule is conjugated to a tracer enzyme or coating protein to set up the assay. Here we report the use of peptides that mimic the analyte as advantageous substitutes of competing haptens. These peptides, which open opportunities for innovation in the development of tracer reagents, can be selected from phage display libraries in a straightforward systematic manner. The concept was proven using assays for the herbicides molinate and atrazine as model systems. Several characteristics of the selection process that may affect the final assay were analyzed, such as the phage coat proteins fused to the peptide, the use of linear or constrained peptide libraries, the effect of the concentration of analyte used during the selection process, and the use of monoclonal or polyclonal antibodies as selector molecules. In all cases we found that the selected peptides performed with improved sensitivity as compared with the chemical hapten conventional assays, showing an analogous cross-reactivity pattern. Interestingly, the phage particles perform as robust and highly standardized assay reagents, and due to their filamentous repetitive structure, they function as sensitive multienzymatic reporters.

Amino Acid Sequence↗
PubMedDate not supplied

Selection of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a phage display peptide library.

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against alpha-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with alpha-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of alpha-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for alpha-chymotrypsin was estimated to be 10(-6) M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified alpha-chymotrypsin than for natural alpha-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to alpha-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe- Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of alpha-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.

Amino Acid Sequence↗
PubMed2002

Application of phage display peptide library to autoimmune diabetes: identification of IA-2/ICA512bdc dominant autoantigenic epitopes.

Autoantigenic epitope mapping represents a critical issue in autoimmune diseases. The islet tyrosine phosphatase-like protein IA-2/ICA512bdc is a major autoantigen in type 1 diabetes (IDDM), but the epitopes responsible for autoantibody binding have been only partially defined. The aim of our study was to identify ICA512bdc epitopes, and in particular mini-epitopes, utilizing a novel strategy for autoimmune diseases. The study was performed in three sequential steps: (1) construction of a lambda-phage surface-displayed ICA512bdc cDNA library with the methodology of tagged random priming with peptides displayed as a fusion to the C terminus of the capsid protein D; (2) affinity selection of the resulting library, followed by immunoscreening, enzyme-linked immunosorbent assay and sequence analysis of positive clones, and (3) radioimmunoprecipitation to detect autoantibodies to the selected clones. This strategy resulted in the identification of two epitopes (IA-2 residues 761 - 964 and 929 - 979), which were recognized by 100 % and 62.9 % ICA512bdc-positive IDDM patients, respectively. Interestingly, the larger clone was detected also by a proportion (16.7 %) of new onset ICA512bdc-negative patients, thus suggesting that this region contains not only the main autoantigenic repertoire of ICA512bdc molecule, but is able to detect IA-2 autoantibodies in even higher percentages of patients. In addition, this study showed the existence of multiple epitopes located in the C-terminal domain of the IA-2 protein, one of which is formed by the 50 C-terminal amino acids, and provided evidence that the strategy used represents a valid tool for identification of epitopes within autoantigenic molecules.

Adolescent↗
PubMed1995

Structural characterization of peptides that bind synovial fluid antibodies from RA patients: a novel strategy for identification of disease-related epitopes using a random peptide library.

Phage epitope library technology has become a powerful tool for identifying determinants recognized by homogenous antibodies. Screening of human sera or fluids with random peptide phage libraries is a novel approach that can dissect common features at the amino acid level in individuals infected by the same etiological agent(s). In this study we have analyzed the specificity of antibodies in synovial fluids (SF) obtained from patients with rheumatoid arthritis (RA). We found that a high proportion of the peptides binding SF antibodies differ from those binding serum antibodies, suggesting that synovial B cells are expanded as a result of local antigen stimulation. When all the selected phages (enriched library) that bind the SF antibodies from a seropositive RA patient were further screened by the use of different SF antibodies with or without rheumatoid factor activity, we identified common SF antibody specificities (IRRSETPRA, RRRVRNSDT, PVSSTRGNM). Antibodies against these common specificities are also found in SF from other RA patients. Taken together, the present results open the possibility of defining the entire set of synovium antibody specificities and also common SF specificities between RA patients.

Amino Acid Sequence↗
PubMed1998

Targeting growth factor and cytokine receptors with recombinant peptide libraries.

The single-transmembrane-spanning receptors of cytokines and growth factors have historically proven resistant to the small-molecule screening efforts of the pharmaceutical industry. Advances in combinatorial library approaches to ligand discovery have begun to show success with these targets. There are several recent reports of peptides, derived from randomly assembled collections of L-peptides expressed in recombinant display vectors, that are high-affinity antagonists and even agonists of these receptors. These results indicate that molecules much smaller than the natural protein factors can interact effectively with these receptors, and this may lead the way to the discovery of even smaller nonpeptidic agents.

Amino Acid Sequence↗
PubMed1999

Evaluation of an Epstein-Barr virus (EBV) immunoglobulin M enzyme-linked immunosorbent assay using a synthetic convergent peptide library, or mixotope, for diagnosis of primary EBV infection.

An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive.

Amino Acid Sequence↗
PubMed1993

Generation and screening of an oligonucleotide-encoded synthetic peptide library.

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.

Amino Acid Sequence↗
PubMed1994

Use of an oriented peptide library to determine the optimal substrates of protein kinases.

BACKGROUND: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals. A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly. Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking. RESULTS: We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates. The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity. We also discuss a model incorporating the optimal cyclin B-Cdc2 substrate into the known crystal structure of this kinase. CONCLUSIONS: Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.

Amino Acid Sequence↗