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

• Published in: Journal of microbiology and biotechnology Vol. 28; no. 10; pp. 1749 - 1759
• Main 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
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국미생물·생명공학회 28.10.2018
• Subjects: Animals; Chromatography, Liquid; Cloning, Molecular; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Humans; Inclusion Bodies; Mice; Prion Proteins - biosynthesis; Prion Proteins - chemistry; Prion Proteins - isolation & purification; Prion Proteins - metabolism; Protein Folding; Protein Structure, Secondary; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; 생물학; seeding activity; recombinant prion protein; high-cell density culture; Expression; purification
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1805.05067

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 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 high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.