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 (PrPC). PrPSc 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 PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA.
| Original language | English |
|---|---|
| Article number | e18047 |
| Journal | PLoS One |
| Volume | 6 |
| Issue number | 3 |
| DOIs | |
| State | Published - 2011 |
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All Science Journal Classification (ASJC) codes
- Agricultural and Biological Sciences(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Medicine(all)
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In vitro amplification of misfolded prion protein using lysate of cultured cells. / Mays, Charles E.; Yeom, Jihyun; Kang, Hae Eun; Bian, Jifeng; Khaychuk, Vadim; Kim, Younghwan; Bartz, Jason C.; Telling, Glenn C.; Ryou, Chongsuk.
In: PLoS One, Vol. 6, No. 3, e18047, 2011.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - In vitro amplification of misfolded prion protein using lysate of cultured cells
AU - Mays, Charles E.
AU - Yeom, Jihyun
AU - Kang, Hae Eun
AU - Bian, Jifeng
AU - Khaychuk, Vadim
AU - Kim, Younghwan
AU - Bartz, Jason C.
AU - Telling, Glenn C.
AU - Ryou, Chongsuk
PY - 2011
Y1 - 2011
N2 - 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 (PrPC). PrPSc 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 PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA.
AB - 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 (PrPC). PrPSc 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 PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA.
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U2 - 10.1371/journal.pone.0018047
DO - 10.1371/journal.pone.0018047
M3 - Article
VL - 6
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 3
M1 - e18047
ER -