Expression and purification of human FANCI and FANCD2 using Escherichia coli cells

Daisuke Takahashi, Koichi Sato, Mayo Shimomuki, Minoru Takata, Hitoshi Kurumizaka

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The DNA interstrand crosslink (ICL) is an extremely deleterious DNA lesion that covalently crosslinks complementary strands and prevents the strand-separation reaction. In higher eukaryotes, the Fanconi anemia proteins, FANCI and FANCD2, form a heterodimer and play essential roles in ICL repair. Human FANCI and FANCD2 are large proteins with molecular masses of 149 kDa and 164 kDa, respectively, and were reportedly purified using a baculovirus expression system with insect cells. We have established a novel expression and purification procedure for human FANCD2 and FANCI, using Escherichia coli cells. The human FANCD2 and FANCI proteins purified by this bacterial expression method formed a stable heterodimer, and exhibited DNA binding and histone chaperone activities, as previously reported for the proteins purified by the baculovirus system. Therefore, these purification methods for human FANCI and FANCD2 provide novel procedures to facilitate structural and biochemical studies of human FANCI and FANCD2.

Original languageEnglish
Pages (from-to)8-15
Number of pages8
JournalProtein Expression and Purification
Volume103
DOIs
Publication statusPublished - 2014 Nov

Keywords

  • Bacterial expression
  • DNA interstrand crosslink
  • FANCI-FANCD2 complex
  • Fanconi anemia

ASJC Scopus subject areas

  • Biotechnology

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