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KMID : 0545120180280101749
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 10 p.1749 ~ p.1759
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High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay
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Hwang Hae-Gwang
Kim Dae-Hwan
Lee Jeong-Min
Mo Young-Won
Lee Se-Hoon
Lee Yong-Jin
Hyeon Jae-Wook
Lee Sol-Moe
Cheon Yong-Pil
Choi Eun-Kyoung
Kim Su-Yeon
Lee Yeong-Seon
Son Young-Jin
Ryou Chong-Suk
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Abstract
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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 α-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 highpurity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.
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KEYWORD
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Expression, high-cell density culture, recombinant prion protein, purification, seeding activity
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