Heterologous expression, purification, activity and conformational studies of different forms of dianthin 30.
Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli. Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide. The production of a form of dianthin 30, which includes the pro-signal, is described as well. Both dianthin 30 delta 255-270 and dianthin 30 expressed in E. coli are mainly localized (90%) in the soluble fraction. Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively. Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture. RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious. In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection. These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.
Legname
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G
Department of Immuno-Oncology, Centro Ricerche Italfarmaco S.p.A., Milano, Italy.
Fossati
G
G
Monzini
N
N
Gromo
G
G
Marcucci
F
F
Mascagni
P
P
Modena
D
D
D000998:Antiviral Agents / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D054789:Ribosome Inactivating Proteins, Type 1; D017384:Sequence Deletion
D000998:Antiviral Agents; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D006026:Glycoside Hydrolases