title: Putative prion protein from Fugu (Takifugu rubripes).
abstract: Prion proteins (PrP) of mammals, birds, reptiles and amphibians have been successfully cloned, expressed and purified in sufficient yields to enable 3D structure determination by NMR spectroscopy in solution. More recently, PrP ortholog genes have also been identified in several fish species, based on sequence relationships with tetrapod PrPs. Even though the sequence homology of fish PrPs to tetrapod PrPs is below 25%, structure prediction programs indicate a similar organization of the 3D structure. In this study, we generated recombinant polypeptide constructs that were expected to include the C-terminal folded domain of Fugu-PrP1 and analyzed these proteins using biochemical and biophysical methods. Because soluble expression could not be achieved, and refolding from guanidine-HCl did not result in a properly folded protein, we co-expressed Escherichia coli chaperone proteins in order to obtain the protein in a soluble form. Although CD spectroscopy indicated the presence of some regular secondary structure in the protein thus obtained, there was no evidence for a globular 3D fold in the NMR spectra. We thus conclude that the polypeptide products of the fish genes annotated as corresponding to bona fide prnp genes in non-fish species cannot be prepared for structural studies when using procedures similar to those that were successfully used with PrPs from mammals, birds, reptiles and amphibians.
authors: Institute of Molecular Biology and Biophysics, ETH Zurich, Schafmattstrasse 20, Zurich, Switzerland.
authors: Barbara
authors: B
authors: Christen
authors: Kurt
authors: K
authors: Wüthrich
authors: Simone
authors: S
authors: Hornemann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011328:Prions / Q000737:chemistry / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D023741:Takifugu
chemical_list: D011328:Prions