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

Title
High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay
Authors
Hwang, Hae-GwangKim, Dae-HwanLee, JeongminMo, YoungwonLee, Se-HoonLee, YongjinHyeon, Jae WookLee, Sol MoeCheon, Yong-PilChoi, Eun-KyoungKim, Su YeonLee, Yeong SeonSon, Young-JinRyou, Chongsuk
DGIST Authors
Hwang, Hae-Gwang; Kim, Dae-Hwan; Lee, Jeongmin; Mo, Youngwon; Lee, Se-Hoon; Lee, Yongjin; Hyeon, Jae Wook; Lee, Sol Moe; Cheon, Yong-Pil; Choi, Eun-Kyoung; Kim, Su Yeon; Lee, Yeong Seon; Son, Young-Jin; Ryou, Chongsuk
Issue Date
2018-10
Citation
Journal of Microbiology and Biotechnology, 28(10), 1749-1759
Type
Article
Article Type
Article
Author Keywords
Expressionhigh cell-density culturerecombinant prion proteinpurificationseeding activity
Keywords
MONOCLONAL-ANTIBODIESESCHERICHIA-COLIEXPRESSIONPRPPURIFICATIONSCRAPIECONVERSIONCELLSCONFORMATIONISOFORM
ISSN
1017-7825
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.
URI
http://hdl.handle.net/20.500.11750/9421
DOI
10.4014/jmb.1805.05067
Publisher
한국미생물·생명공학회
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