The master transcription factor of the Plasmodium merozoite formation. [DIP-seq]

To investigate the DNA-binding property of two tandem AP2 domains of PbSIP2, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis were performed. Recombinant AP2 domains fused with maltose-binding protein (MBP) were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using amylose resin. The obtained DNA fragments were sequenced via the next generation sequencing. DNA fragment encoding the AP2 domains of PbSIP2 was cloned into the expression vector pMal-c5X (NEB). Escherichia coli strain DH5α transformed with this plasmid was cultured for 12 h at 37 °C. Next, expression of the MBP-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 AP2 domains fused with MBP was purified using amylose resin, and eluted with 10 mM maltose solution. Subsequently, the MBP-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 amylose 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 maltose 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.