highly efficient protein misfolding cyclic amplification高效的蛋白质错误折叠循环放大.pdfVIP

Highly Efficient Protein Misfolding Cyclic Amplification 1. 1. 1 1 Nuria Gonzalez-Montalban , Natallia Makarava , Valeriy G. Ostapchenko , Regina Savtchenk , Irina Alexeeva2, Robert G. Rohwer2,3, Ilia V. Baskakov1,4* 1 Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America, 2 Medical Research Service, Veterans Affairs Maryland Health Care System, Baltimore, Maryland, United States of America, 3 Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 4 Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America Abstract Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ,10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be