NR AWDG
AU Charveriat,M.; Picoli,C.; Nouvel,V.; Lenuzza,N.; Aubry,F.; Correia,E.; Reboul,M.; Deslys,J.P.; Mouthon,F.
TI A new assay in E. coli to quantify the aggregation state of prion protein mutants
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-06
PT Konferenz-Poster
AB Transmissible spongiform encephalopathies are mainly characterized by the accumulation of the prion protein (PrP) as an abnormal aggregated form. However only few models help to evaluate and quantify this aggregation state in vivo. We present a general method to assess PrP solubility and aggregation. This model is based on the structural complementation between the alpha- and the omega-fragments of the ß-galactosidase enzyme in E. coli. Different constructions of the PrP sequence (full-length protein, or protein lacking the N-terminus, or with extra octa-repeats) are designed, merged in open frame with the ß-fragment sequence and cloned in alpha-omega+ bacteria. These fusions are functional, as they have been sequenced and validated by Western Blot using anti-PrP antibodies. Solubility of the fusions correlates with a high ß-galactosidase activity, and aggregation with a low activity: aggregation or solubility of the fusion-protein is thus directly assayed by the enzyme activity. We have therefore developed three bacterial strains with a clear and quantitative phenotype of the aggregation or solubility of the prion protein: one "fully aggregated" model using the full-length prion protein with 11 octa-repeats, one "partially aggregated" model with the full-length protein and one "soluble" model with an N-terminus deleted PrP sequence. The aggregation can be tested in high-throughput assays, since 96 samples can be tested simultaneously: our method can constitute a new screening test for drugs that could disrupt or prevent the aggregation of the prion protein. Furthermore, we will generate a library of PrP mutants from different species, by random mutagenesis, to investigate PrP aggregation. Thus, our model might provide a new insight in the mechanisms underlying prion formation and in the selection of original drugs.
AD CEA/DSV/DRM/GIDTIP, Fontenay-aux-Roses, France. E-mail: mathieu.charveriat@cea.fr
SP englisch
PO Italien