Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ)

Roopa Dharmatti, Hideyuki Miyatake, Chen Zhang, Xueli Ren, Akiko Yumoto, Daisuke Kiga, Masayuki Yamamura, Yoshihiro Ito

研究成果: Article

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Human folate receptors (hFRα and hFRβ) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requires a large amount of hFRs. However, it is difficult to prepare active forms of hFRs from prokaryotic cells because of their high content of cysteine residues that form disulfide bonds. Here, we prepared active forms of hFRα and hFRβ from inclusion bodies of Escherichia coli. The crucial steps in our preparation were intensive washing of the inclusion bodies to remove impurities derived from E. coli and gradual dropping of solubilized hFRs into refolding buffers to correctly reform disulfide bonds. The binding activity of prepared hFRs to folate was confirmed by biolayer interferometry measurements. Finally, we successfully prepared the active form of 2.52 mg hFRα and 2.4 mg hFRβ from 10 g of E. coli cell bodies.

元の言語English
ページ(範囲)17-22
ページ数6
ジャーナルProtein Expression and Purification
149
DOI
出版物ステータスPublished - 2018 9 1
外部発表Yes

ASJC Scopus subject areas

  • Biotechnology

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