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Updated: Jun 3, 2026
Protein Misfolding Cyclic Amplification of Prions
Published on: November 7, 2012
In vitro amplification of misfolded prion protein using lysate of cultured cells
Charles E Mays1, Jihyun Yeom, Hae-Eun Kang
1Department of Microbiology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America.
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.
Insights
This study introduces a simplified method for prion protein (PrP) amplification using cell lysates, overcoming limitations of traditional brain-derived or purified PrP sources. This advance offers a more accessible and versatile approach for prion research.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Protein misfolding cyclic amplification (PMCA) is crucial for studying prion protein (PrP) conversion.
- Traditional PMCA relies on brain material or purified PrP, facing limitations in availability and preparation time.
- Species-specific PrP mutations or polymorphisms further complicate substrate sourcing.
Purpose of the Study:
- To develop a convenient and versatile PMCA procedure independent of limited substrate sources.
- To explore the use of cell lysates expressing cellular PrP (PrP(C)) as an alternative substrate for PMCA.
- To assess the efficiency of PrP(Sc) amplification using cell lysate-based PMCA.
Main Methods:
- PMCA was performed using lysates from RK13 cells stably transfected with mouse or Syrian hamster PrP genes.
- Lysates from various neuronal and non-neuronal cell lines were tested for PMCA suitability.
- The impact of PrP(C) abundance in cell lysates on PrP(Sc) amplification efficiency was investigated.
Main Results:
- PrP(Sc) was efficiently amplified using lysates from RK13 cells expressing PrP.
- Successful PMCA was achieved with lysates from diverse cell lines, indicating broad applicability.
- High abundance of PrP(C) in cell lysate was identified as a critical factor for efficient PrP(Sc) amplification.
Conclusions:
- Cell lysates expressing abundant PrP(C) serve as an excellent and versatile substrate for PMCA.
- This novel approach simplifies PMCA procedures and expands substrate accessibility for prion research.
- The findings pave the way for more convenient and widespread prion disease studies.
