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. Overall design: DNA fragment encoding the AP2 domains of PbSIP2 was cloned into the expression vector pMal-c5X (NEB). Escherichia coli strain DH5a transformed with this plasmid was cultured for 12 h at 37 °C. Next, expression of the MBP-fused protein was induced by adding isopropyl ß-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.