Protein misfolding cyclic amplification
Protein misfolding cyclic amplification is an amplification technique to multiply misfolded prions originally developed by Soto and colleagues. It is a test for spongiform encephalopathies like chronic wasting disease or bovine spongiform encephalopathy.
Technique
The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into the prion being tested for.Automation
The technology has been automated, leading to a dramatic increase in the efficiency of amplification. Now, a single cycle results in a 2500-fold increase in sensitivity of detection over western blotting, whereas 2 and 7 consecutive cycles result in 6 million and 3 billion-fold increases in sensitivity of detection over western blotting, a technique widely used in BSE surveillance in several countries.Starting material
PMCA was originally based on the normal prion protein from healthy brain tissue, which is expensive. The advent of recombinant proteins have lower the cost somewhat, but the steps required to obtain the pure protein are laborious. In 2011, it was found that simply putting a prion protein transgene into a cell line and then lysing the cell without purification is enough. This is expected to make PMCA much cheaper. The cell line does not need to be of a neuronal origin.PMCA is most easily performed with catalysts which are abundant even in healthy cells: a polyanion and a phospholipid. A cell lysate would provide both of these catalysts and most clumps of PrPSc contain catalyst polyanion molecules anyways. Synthetic versions of these catalysts such as poly RNA and 1-palmitoyl-2-oleoylphosphatidylglycerol also work for propagating PrPSc.
Additional required materials include buffer salts and detergent.
Unseeded PMCA
PMCA can work even without a starting mass of PrPSc. While this behavior is not desirable for those using PMCA as a detection tool, it has implications for understanding the nature of the TSE pathogen. This conversion is analogous to the sporadic form of TSE.- RNA from healthy mouse liver combined with POPG can convert recombinant PrPC made in E. coli into PrPSc in 17 cycles.
- Buffer salts and detergent alone can recombinant Syrian hamster PrPC made in E. coli into PrPSc in 18 cycles.
Uses
Prion proteins
The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrPC and recombinant PrPC with the sole addition of synthetic polyanions and lipids. These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute some of the strongest evidence in favor of the prion hypothesis.PMCA has been applied to replicate the misfolded protein from diverse species. The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrPSc and strikingly it is infectious to wild type animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.