TAILIEUCHUNG - Báo cáo khoa học: Putative prion protein from Fugu (Takifugu rubripes)

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. | ỊFEBS Journal Putative prion protein from Fugu Takifugu rubripes Barbara Christen Kurt Wuthrich and Simone Hornemann Institute of Molecular Biology and Biophysics ETH Zurich Switzerland Keywords chaperone co-expression fish prion protein nuclear magnetic resonance Takifugu rubripes transmissible spongiform encephalopathy Correspondence S. Hornemann Institute of Molecular Biology and Biophysics Schafmattstrasse 20 ETH Zurich CH-8093 Zurich Switzerland Fax 41 44 633 1484 Tel 41 44 633 3453 E-mail Website http groups wuthrich_group Received 24 August 2007 revised 14 November 2007 accepted 16 November 2007 doi 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

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