Homeodomain-like domain of Plasmodium berghei HDP1 binds to TGCACA motif.

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. 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.