Biomedical subjects

Antonella Bandiera

Publications and source records attributed to Antonella Bandiera.

5 recordsLinked to original sources

PubMed2005

In-vitro dual binding activity of a evolutionarily related subgroup of hnRNP proteins.

The wide family of heterogeneous nuclear ribonucleoproteins (hnRNPs) comprises members that interact with single-stranded nucleic acids. On the basis of their structure, some of them are characterised by a tandem RNA-binding domain (RBD) and a glycine-rich C-terminus, showing a high degree of homology. Recently, we have isolated some proteins belonging to this group that interact with single-stranded cytosine-block telomeric DNA. The aim of the present investigation is to better characterise the relationship of some structural features shared by these proteins and their in-vitro interaction with the telomeric type sequences. We analysed the in-vitro binding properties of some of these components toward both single-stranded telomeric motifs. Using deletion mutants, the relationship between cytosine-rich motif binding activity and the structural features of one of these proteins is further characterized. This binding activity appears to be related to a subgroup of the 2xRBD+Glycine rich hnRNP, suggesting functionally distinct properties of these proteins, in agreement with their evolutionary relationship.

Amino Acid Sequence↗

PubMed2005

Vertebrate 2xRBD hnRNP proteins: a comparative analysis of genome, mRNA and protein sequences.

hnRNP proteins are involved in many cell functions, primarily in pre-mRNA processing. We report here a comparative analysis of the genes of the 2xRBD members of the hnRNP family and of their expression products. Starting from the seven well characterized hnRNP members of human and murine origin (A0, A1, A2/B1, A3, AB, D and DL) and the three MuSashI-like proteins with related RBD tandems (MSI1, MSI2 and DAZAP1), we identified through BLAST search 12 homologous genes in the genome of Danio rerio and 10 in the genome of Takifugu rubripes, which can be divided into three subgroups, each with its highly conserved exon/intron structure, matching perfectly the exon/intron structures found in human and mouse genes. An exception is the gene of hnRNP A0, which is intronless consistently in all the four species. The analysis has been supported also at the level of cDNA and EST databases and extended in this respect to other vertebrate species, namely chicken, Xenopus laevis and Silurana tropicalis. PHYLIP 3.62 package (SEQBOOT, PROTDIST/DNADIST, NEIGHBOR, CONSENSE) was used for all the proteins and their CDSs and human RBDs I and II to infer relevant aspects of the phylogenesis of these proteins. Some clues to the evolution of introns in these genes have come from the analysis of their distribution in homologous genes of other eukaryotes, namely Ciona, Drosophila, Caenorhabditis, Saccharomyces and Arabidopsis.

Amino Acid Sequence↗

PubMed2005

Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system.

In this work, the intein fusion approach was used for expression and purification of cathelicidin-like peptide SMAP-29 from Escherichia coli cultures. To overcome the high toxicity of the antimicrobial peptide against host cells, both C- and N-terminal fusions with Sce VMA intein were evaluated. The fusion of SMAP-29 with the N-terminus of intein had a dramatic lethal effect. In contrast, chimeric constructs harboring SMAP-29 linked to the C-terminus of intein displayed no significant inhibition of bacterial growth. Expression of intein-SMAP fusion protein was then induced in ER2566 E. coli strain by IPTG addition and different experimental conditions were tested in order to optimize the recovery of the soluble protein complex. Peptide purification was carried out by affinity chromatography: the chitin binding domain linked to intein was used to immobilize the chimeric protein on a chitin column and intein-mediated splicing of target peptide was obtained by thiol addition. Microbroth dilution assay showed that recombinant SMAP-29 displayed a high, dose-dependent bactericidal activity. These data demonstrate that the fusion of SMAP-29 with C-intein was able to inactivate the antimicrobial properties of the cathelicidin peptide allowing the expression of fusion protein in the host cell. The intein-mediated purification supplied an effective way to recover the fusion partner in its proper biologically active form.

Amino Acid Sequence↗

PubMed2005

Expression and characterization of human-elastin-repeat-based temperature-responsive protein polymers for biotechnological purposes.

Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin-like polypeptides are a well-defined family of polymers with noteworthy characteristic based on the VPGVG repeated motif of bovine elastin. In the human homologue, the most regular sequence is represented by the repetition of the VAPGVG hexapeptidic motif. On the basis of this sequence, a synthetic gene has been designed, cloned and expressed in Escherichia coli to obtain artificial protein polymers. The rapid one-step in-frame cloning of any biologically active sequence can be achieved directly in the expression vector, allowing further improvement of the potential of the resulting product.

Amino Acid Motifs↗

PubMed2003

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines.

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.

Base Sequence↗