Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.

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Production of a soluble and functional recombinant streptavidin in Escherichia coli.

The cDNA for streptavidin (residues 15-159) was subcloned into an expression vector in fusion at the N-terminus with the T7-tag (12 residues). Conditions were found to express the protein in Escherichia coli in a soluble, assembled, and active form. The protein was purified in two simple steps which involved heating at 75 degreesC and affinity chromatography on iminobiotin agarose.

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli.

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins.

Biochemical and immunological characterization of recombinant allergen Lol p 1.

Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract.

Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria.

Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis.

Chromogranin A fragments modulate cell adhesion. Identification and characterization of a pro-adhesive domain.

Although several functions have been suggested for chromogranin A, a glycoprotein secreted by many neuroendocrine cells, the physiological role of this protein and of its proteolytic fragments has not been established. We have found that mixtures of chromogranin A fragments can inhibit fibroblast adhesion. The anti-adhesive activity was converted into pro-adhesive activity by limited trypsin treatment.

Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications.

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain.

Production, purification and characterization of an anti-(carcinoembryonic antigen) recombinant single-chain Fv antibody fragment.

Specific targeting of radioactive agents to tumour cells has been the main objective of the in vivo use of monoclonal antibodies and their fragments. In particular, specific antibodies to carcinoembryonic antigen (CEA)-expressing tumours can be used either for diagnosis or therapy, if targeting could be improved. The expression of antibody fragments in bacteria allows the preparation of engineered molecules with antigen-binding properties and a better penetration into the tumour.

Characterisation of circulating chromogranin A in human cancer patients.

The structure of circulating chromogranin A (CgA) of phaeochromocytoma patients was characterised and compared with that of CgA extracted from tumours. Size exclusion chromatography experiments provided evidence that CgA is present in the blood of different patients, as well as in tumour extracts, as multiple forms having different hydrodynamic sizes of 600 kDa (CgA-I), 100 kDa (CgA-II) and 55 kDA (CgA-III). The amount of each CgA form as a proportion of the total antigenic material was different in different patients.

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.

A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.

EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing.

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.

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306.

Beta-nerve growth factor (beta-NGF) mRNA expression in the parkinsonian adrenal gland.

The adrenal gland is a well-demonstrated source for different neurotrophic factors. The presence of the beta-nerve growth factor (beta-NGF) mRNA in the adrenal tissue used for grafting in a Parkinsonian patient is reported here. Adrenal samples were obtained on the day of implantation, and a specific cDNA was synthesized after the extraction of total RNA using a synthetic oligonucleotide as a reverse transcription primer.

Molecular cloning of an adducin-like protein: evidence of a polymorphism in the normotensive and hypertensive rats of the Milan strain.

Differences genetically associated with the development of hypertension in a strain of genetically hypertensive rat (MHS) were described in ion transport across erythrocyte membranes compared to normotensive control (MNS). Antibodies against the MNS ghost proteins were raised in the MHS, producing an immunoreaction against a 105 KDa protein later identified as adducin. A clone coding for a portion of mouse adducin was isolated with these antibodies.

Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis.

A form of familial hypobetalipoproteinaemia not due to a mutation in the apolipoprotein B gene.

Familial hypobetalipoproteinaemia (FHBL) is a dominant disorder of lipoprotein metabolism characterized by levels of apolipoprotein B-carrying lipoproteins (VLDL, IDL and LDL) which are 50% of the normal levels in the heterozygotes and almost absent in the homozygotes. Several reports have recently shown that the underlying defect in FHBL involves different mutations in the apo B gene which lead to reduced levels of apo B mRNA or to the production of truncated forms of apo B having either a lower synthetic rate or a higher catabolic rate than normal apo B. We here present a three-generation family with several FHBL members in which the linkage analysis shows absence of co-segregation between apo B gene alleles and the hypocholesterolaemic phenotype.

DNA based diagnostic tests: recombinant DNA and cardiovascular disease risk factors.

Advances in molecular biology and medical biotechnology are continuously creating exciting possibilities for DNA based diagnostics. It is now possible by simple procedures to detect polymorphic DNA markers, structural variants and regulatory mutants of human genes, allowing detailed genotyping of patients. The innovative combination of immunoenzymatic techniques, monoclonal antibodies and recombinant tracer proteins, results in new DNA based tests for the determination of important biochemical parameters, in order to define more precisely the phenotype and hence assess the individual risk.

Human apolipoprotein A-I gene promoter polymorphism: association with hyperalphalipoproteinemia.

An apolipoprotein A-I gene promoter polymorphism, due to an adenine (A) to guanine (G) transition 78 base pairs upstream from the transcription initiation site, was studied by amplification of the corresponding region of the apoA-I gene, DNA sequencing, and allele-specific oligonucleotide hybridization. The frequency of the polymorphism was studied on female and male individuals classified into three groups according to the high density lipoprotein (HDL) cholesterol concentration. The allelic frequencies for the A polymorphism were 0.

Detection of beta-nerve growth factor mRNA in the human fetal brain.

Nerve growth factor (NGF) is a trophic molecule recently demonstrated to interact with different structures in the central nervous system. The expression of the beta-NGF mRNA from human fetal cortices at the 15-16th week of gestational age has been demonstrated and quantitated by polymerase chain reaction amplification of the specific cDNA. beta-NGF mRNA expression in the human brain coincides with the period of active differentiation and synaptogenesis, suggesting that the trophic agent plays a role in the cerebral development.

RIECA: an innovative immuno enzymatic competition assay for apolipoproteins AI and B determination.

The authors describe the production, via recombinant DNA technology, of bifunctional polypeptides for immunoenzymatic assays. These molecules contain an apolipoprotein moiety fused to an active beta-galactosidase enzyme, and are used as tracers in competition assays with specific monoclonal antibodies. The final colorimetric result is inversely correlated with the apolipoproteins plasma values.

Erythrocyte adducin differential properties in the normotensive and hypertensive rats of the Milan strain. Characterization of spleen adducin m-RNA.

Previous studies have shown that erythrocytes from the Milan hypertensive strain of rats (MHS) differ from erythrocytes from the control normotensive strain (MNS). These differences are determined within the stem cells, are genetically associated with the development of hypertension, and are similar to those found between the tubular cells of the two strains. Moreover they seem to be dependent upon the presence of the membrane skeleton proteins.