Aims: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrPSc). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease.
Methods and Results: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high‐degradation activity against a scrapie PrPSc. Sequence analysis indicated that this serine‐protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9–10 and 60–70°C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy‐infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrPSc that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD‐1, a previously reported keratinase.
Conclusions: These results indicate that the isolated protease exhibited higher activity for PrPSc degradation compared with other proteases examined.
Significance and Impact of the Study: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrPSc contamination.
Aims: The purpose of this study was to develop an effective method for detecting prion (PrP) antigenic determinants remaining in bovine meat and bone meal (MBM) using pressurized fluid extraction (PSE) equipment and flow microbead immunoassay (FMI).
Methods and Results: Using the FMI, bovine recombinant PrP could be determined quantitatively in the 7 pmol–7 nmol range using anti‐PrP peptide polyclonal antibody‐coupled microbeads and anti‐PrP monoclonal antibody (SAF61) as a detection antibody. PSE extraction at 120°C for 5 min under high pressure was most effective for eluting PrP determinants from bovine MBMs. The FMI was capable of detecting PrP determinants in bovine MBM extracts with high specificity and indicated that the MBMs contained high levels of PrP determinants. This assay was also applied to the detection of PrPSc determinants in bovine MBM spiked with a scrapie‐infected brain at a weight ratio of 50 : 1.
Conclusions: These data indicate that this assay was effective for the specific detection of PrP determinants contained in bovine MBM extracts.
Significance and Impact of the Study: To our knowledge, this is the first report detailing the detection of PrP determinants in bovine MBM. The assay could be applied to securing the safety of bovine MBM.
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