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 language | English |
|---|---|
| Pages (from-to) | 8-15 |
| Number of pages | 8 |
| Journal | Protein Expression and Purification |
| Volume | 103 |
| DOIs | |
| Publication status | Published - 2014 |
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Keywords
- Bacterial expression
- DNA interstrand crosslink
- FANCI-FANCD2 complex
- Fanconi anemia
ASJC Scopus subject areas
- Biotechnology
Cite this
Expression and purification of human FANCI and FANCD2 using Escherichia coli cells. / Takahashi, Daisuke; Sato, Koichi; Shimomuki, Mayo; Takata, Minoru; Kurumizaka, Hitoshi.
In: Protein Expression and Purification, Vol. 103, 2014, p. 8-15.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression and purification of human FANCI and FANCD2 using Escherichia coli cells
AU - Takahashi, Daisuke
AU - Sato, Koichi
AU - Shimomuki, Mayo
AU - Takata, Minoru
AU - Kurumizaka, Hitoshi
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - Bacterial expression
KW - DNA interstrand crosslink
KW - FANCI-FANCD2 complex
KW - Fanconi anemia
UR - http://www.scopus.com/inward/record.url?scp=84908125485&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84908125485&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2014.08.012
DO - 10.1016/j.pep.2014.08.012
M3 - Article
VL - 103
SP - 8
EP - 15
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -