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| Status |
Public on Feb 25, 2024 |
| Title |
Homeodomain-like domain of Plasmodium berghei HDP1 binds to TGCACA motif. |
| Organism |
Plasmodium berghei ANKA |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
To investigate the DNA-binding property of homeodomain-like domain of Plasmodium berghei HDP1, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis were performed. Recombinant homeodomain-like domain fused with glutathione S-transferase were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using glutathione-sepharose resin. The obtained DNA fragments were sequenced via the next generation sequencing.
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| Overall design |
DNA fragment encoding the homeodomain of HDP1 was cloned into the expression vector pGEX-6P-1 (Cytiva). Escherichia coli strain DH5α transformed with this plasmid was cultured for 12 h at 37 °C. Next, expression of the GST-fused protein was induced by adding isopropyl β-D-thiogalactopyranoside (final concentration of 200 nM) in the culture and incubating for 9 h at 25 °C. Recombinant homeodomain fused with GST was purified using glutathione-sepharose 4B resin (Cytiva), and eluted with 10 mM glutathione solution. Subsequently, the GST-fused homeodomain was incubated with P. berghei ANKA genomic DNA fragments in binding/washing buffer (10 μM ZnSO4, 2 mM MgCl2, 2 mM Tris-HCl at pH 7.4, 100 mM KCl, and 10% glycerol) for 30 min. The protein/DNA solution was further mixed with glutathione-sepharose resin and incubated for 30 min. After incubation, the resin was washed three times with binding/washing buffer, and bound protein-DNA complexes were eluted with 10 mM glutathione solution. A sequencing library was prepared from the DNA fragments and sequenced using Illumina NextSeq. Before their use for DIP, genomic DNA fragments were also sequenced as an input.
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| Web link |
https://pubmed.ncbi.nlm.nih.gov/38554111/
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| Contributor(s) |
Nishi T, Kaneko I, Iwanaga S, Yuda M |
| Citation(s) |
38554111 |
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| Submission date |
Feb 20, 2024 |
| Last update date |
May 27, 2024 |
| Contact name |
masao yuda |
| E-mail(s) |
m-yuda@doc.medic.mie-u.ac.jp
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| Organization name |
mie university
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| Street address |
edobashi 2-174
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| City |
tsu |
| State/province |
mie |
| ZIP/Postal code |
514-0001 |
| Country |
Japan |
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| Platforms (1) |
| GPL33514 |
DNBSEQ-G400 (Plasmodium berghei ANKA) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE235412 |
Chromatin remodeling events associated with gARID are essential for gametocyte development in Plasmodium |
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| Relations |
| BioProject |
PRJNA1078229 |
