A central issue in the generation of prions from recombinant protein has been the apparently low titers of amyloid fibers formed 31
, which results in long incubation times and often the inability to infect wild-type animals directly. If long incubation times do in fact result from low titers, then this implies that synthetic prion preparations are heterogeneous mixtures containing many different conformations (which limits their usefulness for determining the structure of PrPSc
). Alternatively, long incubation times may indicate that amyloid fibers require maturation or further adaptation of their conformation in order to become fully infectious.
Several laboratories have succeeded in creating prion infectivity under conditions developed for PMCA 4, 32,33
. The initial report for the spontaneous generation of infectivity came in the form of unexpected amplification of prions in negative controls during studies that aimed to identify minimal components necessary for prion amplification in vitro
. The components required for amplification included polyanions in addition to PrPC
, which was accompanied by co-purified lipids. These results were verified by repeating the experiment in a laboratory that had never been used for prion research, reducing the potential for contamination. It was later shown that prions can be generated in a similar fashion using brain homogenate as the substrate, rather than minimal components, and that the newly created prion strain was distinct from a commonly used lab strain 32
. Prions created in these studies using either PrPC
or normal brain homogenate had titers that were sufficient to infect hamsters with incubation periods of 113 to 168 days, while naturally occurring hamster prion strains can have incubation periods of 60–300 days 27, 34
Synthesis of highly infectious prions from recombinant PrP has recently been reported using sonication in the presence of lipids and RNA 4
. The infectivity of these preparations was comparable to naturally occurring strains, implying that the authors have achieved a level of purity several orders of magnitude higher than previously reported. Such high titers may be rapidly detected by bioassay, so the robustness and reproducibility of these findings shall soon become apparent. Despite this remarkable report, the possibility of contamination in PMCA cannot be eliminated — especially given reports of a billion-fold amplification of prion titers using PMCA 22
— and no convincing control has been devised to do so. Even if this finding was attributable to contamination of the starting materials, the ability to amplify prions efficiently using recombinant protein is a significant breakthrough, as it may enable the use of labeled recombinant protein for structural studies.
In related work, recombinant PrP (rather than PrPC
contained in brain homogenates) has been employed with purified PrPSc
seed, obtained from brain homogenate, to initiate the PMCA reaction 35
. This study showed that polyanions and lipids are not required for prion amplification, although trace quantities of such cofactors in the brain-derived seed were not ruled out. In bioassays, the attack rate (i.e., the fraction of inoculated animals that developed prion disease) was consistent with low titers in these preparations, rather than a prion strain with a long-incubation period. Upon serial passage, the incubation periods and biochemistry resulting from these amplified prions were consistent with the naturally strain used as seed, though the pattern of brain lesions indicated some differences. If a novel prion strain was in fact created, this argues that prolonged sonication can alter the conformation of PrPSc
Despite the exciting results, some caveats should be noted about the PMCA approach. This technique results in uneven amplification of prions — from well to well and from experiment to experiment — which produces great variability 36
and prevents the resulting data from being quantified. It is well documented that sonication causes an uneven distribution of energy, resulting in cavitation and high temperatures associated with cavitation 37
as well as the generation of free radicals 38
. Protein conformation is sensitive to temperature denaturation, and free radicals may covalently alter proteins. Both cavitation and free-radical modification of proteins are stochastic processes and inherently difficult to control, potentially explaining the variability observed in PMCA experiments. In contrast, the denaturing agents urea and guanidine, used in the production of synthetic prions elsewhere 2, 3, 5
, result in comparatively even and well defined denaturation of protein throughout the solution. In some cases, sonication initiates amyloid formation with proteins that are usually monomeric 39
, indicating denaturation. Furthermore, such alterations in protein conformation may result in the degradation of the protein 40
. It is noteworthy that the durations of sonication used for PMCA greatly exceed those used in recombinant protein production, often by a factor of over 100. Degradation and denaturation may thus limit the usefulness of products produced by PMCA for structural studies.