title: In vitro amplification of misfolded prion protein using lysate of cultured cells.
abstract: Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrP(C)). PrP(Sc) was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrP(C) in cell lysate was a critical factor to drive efficient PrP(Sc) amplification, our results demonstrate that cell lysate in which PrP(C) is present abundantly serves as an excellent substrate source for PMCA.
authors.affiliation: Department of Microbiology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Mays
authors.forename: Jihyun
authors.initials: J
authors.lastname: Yeom
authors.forename: Hae-Eun
authors.initials: HE
authors.lastname: Kang
authors.forename: Jifeng
authors.initials: J
authors.lastname: Bian
authors.forename: Vadim
authors.initials: V
authors.lastname: Khaychuk
authors.forename: Younghwan
authors.initials: Y
authors.lastname: Kim
authors.forename: Jason C
authors.initials: JC
authors.lastname: Bartz
authors.forename: Glenn C
authors.initials: GC
authors.lastname: Telling
authors.forename: Chongsuk
authors.initials: C
authors.lastname: Ryou
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002457:Cell Extracts / Q000032:analysis*; D002478:Cells, Cultured; D006224:Cricetinae; D051379:Mice; D009474:Neurons / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D018620:PrPSc Proteins / Q000378:metabolism; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011817:Rabbits
chemical_list: D002457:Cell Extracts; D018620:PrPSc Proteins; D011328:Prions