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Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses. [ABSTRACT FROM AUTHOR]

Focuses on profilin as a central regulatory component of the cytoskeleton. Isolation of profilin from trophozoites of the pathogenic Entamoeba histolytica strain SFL-3; Amino acid sequence; Actin-binding protein; Circular DNA cloning.

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding m these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor aI, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3′-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3′-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor aI. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor aI isoforms revealed identifies of 96–99%. The sequence identities between the Cor aI isoforms and Bet vI, the major birch pollen allergen, were 71–73% (80.5–83% similarity). Comparing amino acid sequences of Cor aI isoforms with the published sequences of Aln gI, the major allergen from alder, and Car bI and isoforms, the major allergen from hornbeam, 75.5–76.7% identity (83.6–85% similarity) and 83.6–89.9% sequence identity (89.3–95% similarity), respectively, was found. The four Cor aI cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor aI. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet vI) mAb, BIP 1. Our results demonstrate that Cor aI isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies. [ABSTRACT FROM AUTHOR]

The 43–kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N–terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex–allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 μg/ml in skin prick tests produced wheal–and–flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS–polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing. [Copyright &y& Elsevier]