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http://dx.doi.org/10.4014/jmb.1805.05067

High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay  

Hwang, Hae-Gwang (Department of Pharmacy, Sunchon National University)
Kim, Dae-Hwan (Department of Pharmacy and Institute of Pharmaceutical Science & Technology, Hanyang University)
Lee, Jeongmin (Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention)
Mo, Youngwon (Department of Pharmacy and Institute of Pharmaceutical Science & Technology, Hanyang University)
Lee, Se-Hoon (Department of Pharmacy, Sunchon National University)
Lee, Yongjin (Department of Pharmacy, Sunchon National University)
Hyeon, Jae Wook (Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention)
Lee, Sol Moe (Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention)
Cheon, Yong-Pil (School of Biological Sciences and Chemistry, Sungshin Women's University)
Choi, Eun-Kyoung (Ilsong Institute of Life Science, Hallym University)
Kim, Su Yeon (Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention)
Lee, Yeong Seon (Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention)
Son, Young-Jin (Department of Pharmacy, Sunchon National University)
Ryou, Chongsuk (Department of Pharmacy and Institute of Pharmaceutical Science & Technology, Hanyang University)
Publication Information
Journal of Microbiology and Biotechnology / v.28, no.10, 2018 , pp. 1749-1759 More about this Journal
Abstract
Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an ${\alpha}$-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.
Keywords
Expression; high cell-density culture; recombinant prion protein; purification; seeding activity;
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