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Updated: Apr 19, 2026
Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System
Published on: July 17, 2021
In vitro amplification of scrapie and chronic wasting disease PrP(res) using baculovirus-expressed recombinant PrP as
Bonto Faburay1, Dongseob Tark, Anumantha G Kanthasamy
1a Department of Diagnostic Medicine and Pathobiology ; College of Veterinary Medicine ; Kansas State University ; Manhattan , KS USA.
Abstract:
Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP(Sc). Studies showed that recombinant cellular PrP, PrP(C), expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP(C) shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP(C) and supports amplification of PrP(Sc) and PrP(CWD) derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP(Sc) or PrP(CWD) production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrP(C) to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.
Insights
Baculovirus-expressed sheep and deer prion protein (PrP) can amplify abnormal prions in vitro without extra co-factors. Adding specific mRNA or manganese enhanced this prion replication process, showing a new substrate for prion disease research.
Area of Science:
- Prion biology and neurodegenerative diseases
- Biochemistry and molecular biology of protein misfolding
- Veterinary medicine and animal health
Background:
- Protein misfolding cyclic amplification (PMCA) mimics prion replication in vitro.
- Recombinant PrP(C) from E. coli is unsuitable due to lack of glycosylation and GPI anchor.
- Host factors like RNA and divalent cations influence prion disease pathogenesis and PMCA sensitivity.
Purpose of the Study:
- To evaluate baculovirus-expressed sheep and deer PrP genotypes as substrates for seeded PMCA.
- To assess the impact of prion-specific mRNA and manganese on PMCA efficiency.
- To determine if recombinant PrP(C) can support prion amplification without exogenous co-factors.
Main Methods:
- Expression of sheep and white-tailed deer PRNP genotypes using the baculovirus system.
- Seeded PMCA reactions using recombinant PrP(C) as substrate.
- Assessment of conversion efficiency with and without added PrP mRNA and manganese chloride (MnCl2).
Main Results:
- Baculovirus-expressed recombinant PrP(C) exhibited glycoform and GPI-anchor profiles similar to native PrP(C).
- Supported amplification of PrP(Sc) and PrP(CWD) in a single PMCA round without co-factors.
- Species-specific PrP mRNA stimulated conversion efficiency; MnCl2 induced conversion in unseeded PMCA.
Conclusions:
- Baculovirus-expressed sheep and deer PrP are suitable substrates for PMCA, recapitulating prion conversion.
- This system functions effectively without additional exogenous co-factors, simplifying in vitro prion replication studies.
- The findings offer a valuable tool for studying prion diseases in sheep and deer and developing diagnostic methods.
