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Your search keyword '"Tagliabue R"' showing total 3 results
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3 results on '"Tagliabue R"'

1. Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

Author

Mistry AR, Falciola L, Monaco L, Tagliabue R, Acerbis G, Knight A, Harbottle RP, Soria M, Bianchi ME, Coutelle C, and Hart SL

Subjects
Amino Acid Sequence, Animals, Base Sequence, Blotting, Southern, Blotting, Western, Caco-2 Cells, Cloning, Molecular, DNA-Binding Proteins genetics, DNA-Binding Proteins isolation & purification, Gene Dosage, Gene Expression, Gene Transfer Techniques, Genes, Reporter, Genetic Vectors, High Mobility Group Proteins biosynthesis, High Mobility Group Proteins isolation & purification, High Mobility Group Proteins metabolism, Humans, Luciferases genetics, Luciferases metabolism, Molecular Sequence Data, Plasmids, Polymerase Chain Reaction, Protein Binding, Rats, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, High Mobility Group Proteins genetics, Pichia genetics, Recombinant Proteins genetics, Transfection methods
Abstract

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Published
1997
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2. An in vitro amplification approach for the expression of recombinant proteins in mammalian cells

Author

Lucia Monaco, Tagliabue R, Soria MR, and Uhlèn M

Subjects
Chloramphenicol O-Acetyltransferase, Base Sequence, Endothelin-1, Genome, Human, Endothelins, Molecular Sequence Data, Transfection, Muscle, Smooth, Vascular, Recombinant Proteins, Cell Line, Clone Cells, Blotting, Southern, Biopolymers, Humans, Protein Precursors, Nucleic Acid Amplification Techniques, HeLa Cells
Abstract

A method for the expression of recombinant DNA products in mammalian cells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interest are mixed at different molar ratios, and linear polymers are formed using forced head-to-tail ligation. After introduction of the polymers into mammalian cells, transformants with various amounts of the amplified gene unit are obtained. For a first characterization of the system, the gene coding for chloramphenicol acetyltransferase (CAT) has been used to produce polymers containing a single neomycin-resistance gene ligated to different numbers of CAT gene units and used for transfection into HeLa cells. All isolated G418 (gentamycin)-resistant cell transformants which received in vitro amplified DNA were found to express high levels of CAT activity in a stable manner. Southern-blot analysis of individual clones revealed multiple copies of the gene integrated head-to-tail in the genome. This system allowed us to express the gene coding for human prepro-endothelin-1 in A617 human vascular-smooth-muscle cells. The recombinant protein was shown to be correctly processed and biologically active endothelin-1.

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

Author

Massimo Caputi, R. Tagliabue, A. Sidoli, Carlos Melo, Francisco E. Baralle, S. Guenzi, Giorgio Casari, Caputi, M, Casari, GIORGIO NEVIO, Guenzi, S, Tagliabue, R, Sidoli, A, Melo, Ca, and Baralle, Fe

Subjects
Recombinant Fusion Proteins, Molecular Sequence Data, Computational biology, Biology, Exon shuffling, Transfection, Polymerase Chain Reaction, Exon, Exon trapping, Genetics, Humans, RNA, Messenger, Enhancer, Exonic splicing silencer, Base Sequence, Alternative splicing, Exons, Blotting, Northern, Fibronectins, Globins, Alternative Splicing, Enhancer Elements, Genetic, Regulatory sequence, RNA splicing, Mutagenesis, Site-Directed, Gene Deletion, HeLa Cells
Abstract

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. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons.

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