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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from
Atsuko Takeuchi1, Mayumi Komiya, Tetsuyuki Kitamoto
1Department of Neurological Science, Tohoku University Graduate School of Medicine, Japan.
Abstract:
In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.
Insights
This study evaluated disinfectant efficacy against misfolded prion proteins using multi-round protein misfolding cyclic amplification (PMCA). Several disinfectants demonstrated high effectiveness, achieving log reduction factors (LRFs) of 5.8-6.1 log, indicating significant prion inactivation.
Area of Science:
- Prion biology and infectious diseases
- Biochemistry and molecular biology
- Disinfection and sterilization technologies
Background:
- Prion diseases are fatal neurodegenerative disorders caused by misfolded prion proteins.
- Effective inactivation of prions is crucial for preventing disease transmission.
- Assessing disinfectant efficacy against prions requires sensitive and reliable methods.
Purpose of the Study:
- To evaluate the efficacy of various disinfectants in reducing protease-resistant prion protein.
- To establish a quantitative method for disinfectant assessment using multi-round protein misfolding cyclic amplification (PMCA).
- To determine the log reduction factors (LRFs) of tested disinfectants against prions.
Main Methods:
- Hamster brains infected with scrapie strain 263K were homogenized and treated with disinfectants or mock conditions.
- Treated samples underwent multi-round PMCA to amplify residual prion seeds.
- Serial dilutions of mock-treated samples established a titration curve for LRF calculation.
Main Results:
- Multi-round PMCA demonstrated a linear correlation between amplification rounds and prion dilution, yielding LRFs of 3.8-7.3 log for mock-treated samples.
- NaOH treatments (1M and 2M) resulted in LRFs of 6.1 and 5.8 log, respectively.
- Alkaline cleaner and sodium dodecyl sulfate showed LRFs exceeding the evaluation limit (≥7.3 log), indicating very high efficacy.
Conclusions:
- Multi-round PMCA is a valuable tool for assessing disinfectant efficacy against prions.
- Tested disinfectants, including alkaline cleaner and sodium dodecyl sulfate, are highly effective in prion inactivation.
- The established evaluation limit for PMCA is useful for disinfectant assessment and process validation in prion safety.
