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PubMed1999

Identification of multiple HIV-1 CTL epitopes presented by HLA-B*5101 molecules.

We attempted to identify and characterize HIV-1 CTL epitopes presented by HLA-B51 which is associated with a slow progression to AIDS. HLA-B*5101 stabilization assay showed that 33 out of 172 HIV-1 peptides carrying HLA-B*5101 anchor residues bound to HLA-B*5101. Seven peptides were suggested as HIV-1 CTL epitopes presented by HLA-B*5101 because the specific CTL was induced for these peptides in PBMC from three HIV-1 seropositive individuals carrying HLA-B51 by stimulation with HLA-B*5101 binding peptides. Analysis of these epitopes using the specific CTL clones confirmed that six of seven HIV-1 peptides are epitopes presented by HLA-B*5101. Three epitopes presented by HLA-B*5101 are highly conserved among the clade B strain, suggesting that the specific CTL for these epitopes might play an important role in recognition of HIV-1 infected cells. These epitopes will be useful to analyze CTL responses in HIV-1 infected individuals.

Antigen Presentation↗
PubMed2000

Multi-epitope schistosome vaccine candidates tested for protective immunogenicity in mice.

The major challenge in the development of anti-schistosome vaccines is to use defined antigens to stimulate an appropriate immune response that leads to resistance. Several promising candidate vaccine antigens including the glycolytic enzyme triose-phosphate isomerase (SmTPI), a 28 kDa glutathione-S-transferase (Sm28), the myofibrilar protein paramyosin (Sm97), an integral membrane protein (Sm23) and calpain (Smcalpain) have been characterised and their primary sequences derived for Schistosoma mansoni. Furthermore, sequences are available for synthetic peptides mimicking epitopes on these molecules capable of inducing schistosome-specific T- and B-cell responses. These schistosome vaccine candidates have generally been tested with varying degrees of success as single components, with only one report of the use of a multivalent antigen or multi-epitope approach. We describe the assembly of multiple defined and different epitopes of S. mansoni into a variety of single covalent structures; these included a DNA vaccine encoding different epitopes in tandem, the polyprotein itself that is encoded by this DNA and branched synthetic peptide epitope-based polymers in which the individual epitopes are pendant from an inert backbone. Each of the vaccine constructs examined, with the exception of the DNA vaccine, generated antibodies that were capable of binding to a tandem sequence of the epitopes. Although these results were encouraging, none of the constructs protected animals from subsequent challenge infection, indicating that the immune responses elicited were inadequate or inappropriate for parasite killing in vivo.

Animals↗
PubMed2001

Identification of CD4+ T cell epitopes on the fusion (F) and attachment (G) proteins of bovine respiratory syncytial virus (BRSV).

To gain insight into the antigenic structure of the F and G proteins of BRSV, we have mapped CD4+ T cell epitopes on these proteins using synthetic peptides and lymphocytes from vaccinated, naturally infected or experimentally infected calves, in proliferation assays. Bovine CD4+ T cells recognised epitopes that were distributed predominantly within the F1 subunit of the F protein, some of which were adjacent to previously identified B cell epitopes. Bovine CD4+ T cell epitopes within the G protein were mainly located within the cytoplasmic tail. Several immunodominant bovine T cell epitopes within the F protein, that were recognised by calves with different haplotypes, are also recognised by human T cells. Thus, cattle and humans appear to recognise similar T cell epitopes on the F protein. Studies using antibodies to bovine MHC class II and BoLA DR-transfected CHO cells as antigen-presenting cells indicated that immunodominant regions of the F and G proteins contained both DR- and DQ-restricted epitopes. The finding that there was little recognition of the extracellular domain of the G protein by T cells has important implications for vaccine design based on the soluble form of this protein.

Animals↗
PubMed2002

Induction of HLA-A2-restricted CTL responses by a tubular structure carrying human melanoma epitopes.

Epitope-based vaccination strategies designed to induce strong tumor-specific CD8(+) T cell responses are being widely considered for cancer immunotherapy. Here, two recombinant tubular structures, NS1-Mela 1 and NS1-Mela 2, carrying, respectively two HLA-A2 epitopes derived from human melanoma antigens were constructed and their capability to induce CTL responses in vivo were studied in HLA-A2 transgenic mice. Strong CTL responses specific for GnT-V/NA 17-A and gp100 (154-162) epitopes were generated in HLA-A2 transgenic mice immunized by the construct NS1-Mela l carrying these two epitopes. The second construct NS1-Mela 2 carrying both Tyrosinase (369-377Da) and Melan-A/Mart-1 (27-35) epitopes induced a weak Tyrosinase-specific CTL response in mice but failed to induce specific CTL responses against the Melan-A/Mart-1 (27-35) epitope in the tested mice. Thus, recombinant tubular structures containing multiple tumoral epitopes may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Amino Acid Sequence↗
PubMed1997

Synthesis of alpha-gal epitopes on influenza virus vaccines, by recombinant alpha 1,3galactosyltransferase, enables the formation of immune complexes with the natural anti-Gal antibody.

Synthesis of the carbohydrate structure Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-gal epitope) on viral glycoproteins is of interest because of the large amounts of natural antibody (anti-Gal) produced in humans against this epitope. The presence of alpha-gal epitopes on inactivated virus or subviral vaccines is likely to enhance vaccine immunogenicity through in vivo complexing with anti-Gal and the subsequent targeting of the vaccine to Fcy receptors on antigen presenting cells. Our previous studies have demonstrated the increased in vitro immunogenicity of inactivated influenza virus complexed with the anti-Gal antibody. Here we demonstrate a method for engineering the expression of alpha-gal epitopes on influenza virus hemagglutinin (HA) by recombinant alpha 1,3galactosyltransferase (r alpha 1,3GT). We further demonstrate the formation of immune complexes between this de novo synthesized epitope and anti-Gal. HA has multiple N-acetyllactosamine structures which serve as excellent acceptors for r alpha 1,3GT. The luminal portion of marmoset alpha 1,3GT cDNA was produced in large amounts in the baculo virus system and isolated by affinity chromatography on nickel-Sepharose columns. r alpha 1,3GT effectively transferred galactose from UDP-Gal to the N-acetyllactosamine residues of HA on the intact virion or to isolated HA molecules. At least 3000 alpha-gal epitopes were de novo synthesized per virion. The natural anti-Gal antibody bound to these epitopes in ELISA, in western blots and in solution, forming distinct immune complexes. These data suggest that in vivo administration of such vaccines will result in their complexing with anti-Gal, and thus may lead to their increased immunogenicity.

Animals↗
PubMed1998

Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses.

Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.

Adjuvants, Immunologic↗
PubMed2003

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads.

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII.

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope.

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.

Amino Acid Sequence↗
PubMed2006

Membrane topology of human ASBT (SLC10A2) determined by dual label epitope insertion scanning mutagenesis. New evidence for seven transmembrane domains.

The membrane topology of the human apical sodium-dependent bile acid transporter (hASBT) remains unresolved. Whereas N-glycosylation analysis favors a 7 transmembrane (TM) model, membrane insertion scanning supports a 9TM topology. In order to resolve this controversy, we used dual label epitope insertion to systematically examine the topological framework of hASBT. Two distinct epitopes, hemagglutinin (HA) and FLAG, were individually inserted by inverted PCR mutagenesis at strategic positions along the hASBT sequence. Cell surface biotinylation and immunoblotting with epitope-specific and anti-hASBT antibodies confirmed expression and trafficking of the mutants to the plasma membrane. Confocal microscopy confirmed membrane localization of epitope-tagged hASBT in saponin-treated (permeabilized) and nonpermeabilized transfected COS-1 and MDCK cells. Tags at positions 116, 120, 186, 270, and 284 were accessible to the epitope antibodies in nonpermeabilized cells, indicative of the extracellular localization of loops 1 (99-130), 2 (180-191), and 3 (253-287). The corresponding positions in the 9TM model were predicted to be intracellular or membrane bound. Epitope mutants at residues 56, 92, 156, and 221 were only detected after treatment with saponin, indicating the intracellular localizations of loops 1 (50-73), 2 (150-160), 3 (215-227) as predicted by a 7TM model. Our results also confirm the exofacial and cytosolic localization of N- and C-terminal tails, respectively. With the exception of constructs inserted at position 120, epitope mutants displayed active, sodium-dependent taurocholate uptake. Consequently, our study strongly supports a 7TM topology for hASBT and refutes the previously proposed 9TM model.

Binding Sites↗
PubMed1996

Identification of an epitope in antithrombin appearing on insertion of the reactive-bond loop into the A beta-sheet.

Previous work has shown that insertion of the reactive-bond loop of antithrombin into the main beta-sheet of the inhibitor, the A sheet, leads to exposure of epitopes that are not present in intact antithrombin. Identical epitopes are exposed in antithrombin-proteinase complexes, inferring that the reactive-bond loop is inserted into the A beta-sheet also in these complexes. Loop insertion thus presumably is involved in the mechanism of inhibition of target proteinases. In this work, we have identified a linear epitope in bovine antithrombin that reacts with antibodies specific for loop-inserted forms of the inhibitor. This epitope is a hexapeptide sequence comprising residues 342-347, Glu-Asp-Leu-Phe-Ser-Pro, and is located on the surface of the protein just carboxy-terminal of helix I. The Phe residue of this epitope is highly conserved in members of the serpin superfamily and appears to stabilize the region of the epitope in antithrombin and other serpins by interacting with the protein core. The conformational change involving expansion of the A beta-sheet following insertion of the reactive-bond loop is presumably transmitted through this Phe residue to the epitope region, thereby rendering this region accessible to antibodies.

Amino Acid Sequence↗
PubMed1998

Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein.

The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs. MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters. Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein. To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295). These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16). Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function. The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody. Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly. These results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.

ATP-Binding Cassette Transporters↗
PubMed2005

Common physical-chemical properties correlate with similar structure of the IgE epitopes of peanut allergens.

Although many sequences and linear IgE epitopes of allergenic proteins have been identified and archived in databases, structural and physicochemical discriminators that define their specific properties are lacking. Current bioinformatics tools for predicting the potential allergenicity of a novel protein use methods that were not designed to compare peptides. Novel tools to determine the quantitative sequence and three-dimensional (3D) relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/). These peptide comparison tools are based on five-dimensional physicochemical property (PCP) vectors. Sequences from SDAP proteins similar in their physicochemical properties to known epitopes of Ara h 1 and Ara h 2 were identified by calculating property distance (PD) values. A 3D model of Ara h 1 was generated to visualize the 3D structure and surface exposure of the epitope regions and peptides with a low PD value to them. Many sequences similar to the known epitopes were identified in related nut allergens, and others were within the sequences of Ara h 1 and Ara h 2. Some of the sequences with low PD values correspond to other known epitopes. Regions with low PD values to one another in Ara h 1 had similar predicted structure, on opposite sides of the internal dimer axis. The PD scale detected epitope pairs that are similar in structure and/or reactivity with patient IgE. The high immunogenicity and IgE reactivity of peanut allergen proteins might be due to the proteins' arrays of similar antigenic regions on opposite sides of a single protein structure.

2S Albumins, Plant↗
PubMed2003

Binding and cytotoxicity of HPMA copolymer conjugates to lymphocytes mediated by receptor-binding epitopes.

PURPOSE: Studies on the recognition of epitopes presented on a template peptide showed the potential of nonapeptide-related sequences to act as biorecognition sites for the B-cell CD21 receptor. This study was intended to evaluate the capability of three epitope sequences to mediate specific cell binding and to enhance the cytotoxicity of HPMA copolymer conjugates. METHODS: HPMA copolymer conjugates were synthesized containing three different epitopes at various contents and either a fluorescent marker or doxorubicin (DOX). The binding and cytotoxicity of the conjugates to CD21+ Raji B cells and CD21- HSB-2 T cells were evaluated. RESULTS: The epitope-containing conjugates were found to bind to Raji cells at different apparent affinities depending on epitope structure and content. The conjugates generally possessed higher affinities for Raji cells than for HSB-2 cells. Targeted HPMA copolymer-DOX conjugates exhibited higher cytotoxicities than the nontargeted conjugate, likely indicative of enhanced internalization by receptor-mediated endocytosis. HSB-2 cells were more sensitive to both free and polymer-bound DOX than Raji cells; however, the enhancement of cytotoxicity of the conjugates by incorporation of epitopes was more pronounced for Raji cells. CONCLUSIONS: The results verified the concept of using receptor-binding epitopes as targeting moieties in HPMA copolymer conjugates for the delivery of anticancer drugs to lymphoma cells.

Antineoplastic Agents↗
PubMed1997

Tandem peptide epitopes facilitate CD4-dependent activation of T cell clones.

Peptides that consist of two tandemly repeated epitopes joined by a flexible linker have an increased affinity for class II molecules and are more potent at inducing proliferation of T cell clones than monomeric epitopes. The increase in potency of peptides with two epitopes for individual T cell clones is proportional to the relative CD4 dependence of the clones. We show that epitope dimers activate T cell clones that respond sub-optimally to monomeric epitopes presented by APC from HIV-infected donors. We hypothesize that HIV+ APC normally fail to stimulate the clones because virally encoded gp 120 sequesters CD4 from the activation complex, but epitope dimers overcome this effect because they are better able to recruit CD4. The alpha beta heterodimer of human class II (HLA-DR1) is further ordered as a dimer of heterodimers (superdimer) at least in its crystal form. Since class II molecules have an open-ended antigen binding groove, the superdimer is theoretically permissive of stable binding of two peptide epitopes linked in tandem. Our data support a role for the MHC class II dimer of heterodimers in amplifying the proliferative response of T cells to antigen by dint of the superdimers having a higher affinity for CD4 than the nominal class II alpha beta heterodimers.

Antigen-Presenting Cells↗
PubMed1996

Goodpasture's epitope in development of experimental autoimmune glomerulonephritis in rats.

The Goodpasture's epitope (GP) has recently been localized to the last 36 AA of the non-collagenous (NCl) domain of the alpha 3 chain of type IV collagen [alpha 3(IV)]. Since alpha 3(IV) induces glomerulonephritis (GN) in rats and rabbits, the purpose of the present study was to determine if the GP epitope itself could induce GN. We immunized rats with synthetic peptides of GP epitope, 36-mer, alone or as protein conjugates. Rats immunized with bovine GBM served as positive controls. Peptide immunized rats developed high titer antibodies to peptides, but only unconjugated 36-mer induced antibody against human and bovine GBM, but not to rat GBM. Acidic residues and the full length 36-mer were important in production of GBM reactive antibodies. Positive controls developed antibody to GBM without reactivity against 36-mer, had IgG and fibrin on the basement membrane, GN and proteinuria. Kidney eluted antibody was reactive with rat, bovine, and human GBM but not 36-mer. GN rat lymphocytes underwent blast transformation to GBM but not peptide, and peptide immunized animals responded only to the respective peptides. None of the animals immunized with GP peptide epitope, despite the development of anti-peptide antibodies or anti-GBM antibodies, developed any in vivo fixation of antibody to the GBM, abnormal proteinuria, or GN. The present study shows that the GP epitope is sufficient to induce an immune response to the epitope, but it is not sufficient to induce GN. This demonstrates that other factors or epitopes are important in the pathogenicity of GBM induced GN in this model. These remain to be delineated.

Animals↗
PubMed1996

Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.

Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Efficient epitope mapping by bacteriophage lambda surface display.

A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Amino Acid Sequence↗