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E Tamborini

Publications and source records attributed to E Tamborini.

24 records · Page 2Linked to original sources
PubMed1995

Recombinant allergen Lol p II: expression, purification and characterization.

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

Allergens↗
PubMed1994

The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers.

Mammalian ferritins are 24-meric proteins composed of variable proportions of H and L-subunits. The L-chain, in contrast to the H-chain, lacks detectable ferroxidase activity, and its role in ferritin iron incorporation is unclear. In this study, apoferritins were subjected to iron loading with large iron increments to favour spontaneous iron hydrolysis. The homopolymers of the wild-type H-chain, and of a mutant H-chain with an inactivated ferroxidase centre, formed massive protein aggregates, while the L-chain homopolymers remained mostly soluble. The difference between H and L-ferritins was not related to the rate of iron oxidation or to the presence of preformed iron cores. Heteropolymers were constructed in vitro by co-renaturing different proportions of the H-chain with the L-chain or mutant H-chain with an inactivated ferroxidase centre. After loading with high iron increments, protein aggregation of the heteropolymers was reduced when the L-chain content was above 70 to 80%, either in combination with the wild-type H-chain or with the inactivated mutant H-chain. Under acidic conditions (pH 5.5, 1000 Fe atoms per molecule) the heteropolymers with about 20% H and 80% L-chains incorporated three to fourfold more iron into soluble 24-mers than the homopolymers. The data indicate that ferritins with more than 18 L-chains per molecule have the capacity to lower non-specific iron hydrolysis in bulk solution. This property is possibly due to a specific attraction of the incoming oxidized iron into the cavity and may be related to an effect of the L-chain on the cavity microenvironment. It is concluded that under high iron increments the ferritins with high L:H-chain ratios are the most efficient in incorporating iron, and this goes some way to explain why iron storage tissues contain L-rich isoferritins.

Ceruloplasmin↗
PubMed1993

Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II.

The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.

Allergens↗
PubMed1992

DNA methylation of coding and non-coding regions of the human H-RAS gene in normal and tumor tissues.

To study a possible role of DNA methylation in the regulation of expression of the human H-RAS gene in vivo, 41 samples of different normal tissues and 33 tumors of various histotypes were analysed for DNA methylation. In a subset of normal tissues the RNA expression was also examined. The promoter region of the gene showed the features of a CpG island being CpG rich and unmethylated in all the normal tissues and tumors. The coding region displayed an intermediate level of methylation in the majority of normal and tumor tissues. Among the normal tissues only thymus DNA was found to be hypermethylated, while testes and sperm cells DNAs were hypomethylated. The 3' region was found to be completely methylated only in sperm cells and was never completely demethylated. The majority of the tissues showed an intermediate level of methylation, and a certain tissue specificity emerged by comparing tissues of the same and different histotypes. However, the overall methylation of both coding and 3' regions did not correlate with the levels of tissue-specific RNA expression. In heterozygous individuals, allelic methylation of the 3' region showed either perfectly balanced or slightly unbalanced methylation of the two alleles in normal tissues, while an allele-specific methylation was found in 5 out of 12 fresh tumor samples.

Alleles↗