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

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)17-22
Number of pages6
JournalProtein Expression and Purification
Volume149
DOIs
Publication statusPublished - 2018 Sep 1
Externally publishedYes

Fingerprint

Folic Acid
Escherichia coli
Inclusion Bodies
Disulfides
Prokaryotic Cells
Interferometry
Glycosylphosphatidylinositols
Purines
DNA Methylation
Drug Delivery Systems
Cysteine
Buffers
Membrane Proteins
Lipids
Growth

Keywords

  • Bio-layer interferometry (BLI)
  • Escherichia coli expression
  • Folate
  • Gradual dropping refolding
  • Human folate receptor α (hFRα)
  • Human folate receptor β (hFRβ)
  • Immobilized metal affinity chromatography (IMAC)
  • Inclusion body
  • Intensive washing of inclusion body
  • Solubilization

ASJC Scopus subject areas

  • Biotechnology

Cite this

Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ). / Dharmatti, Roopa; Miyatake, Hideyuki; Zhang, Chen; Ren, Xueli; Yumoto, Akiko; Kiga, Daisuke; Yamamura, Masayuki; Ito, Yoshihiro.

In: Protein Expression and Purification, Vol. 149, 01.09.2018, p. 17-22.

Research output: Contribution to journalArticle

Dharmatti, Roopa ; Miyatake, Hideyuki ; Zhang, Chen ; Ren, Xueli ; Yumoto, Akiko ; Kiga, Daisuke ; Yamamura, Masayuki ; Ito, Yoshihiro. / Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ). In: Protein Expression and Purification. 2018 ; Vol. 149. pp. 17-22.
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