DBD region - replicate 1
The coding sequence of DBD region was codon-optimized for Escherichia coli expression and cloned into pET28a with an N-terminal His-Flag tandem tag. The recombinant plasmid was transformed into E. coliBL21(DE3) competent cells. Cultures were grown at 37 C until an OD of 0.5 was reached, at which point protein expression was induced with 0.2 mM isopropyl -D-1-thiogalactopyranoside (IPTG) at 16 C for 18 h. Cells were harvested by centrifugation, lysed by sonication, and the soluble fraction was subjected to Ni-NTA affinity chromatography. Protein purity was assessed by SDS-PAGE and estimated to be >85%. Purified protein was stored in TBS supplemented with 10% glycerol at 80 C. HT-SELEX was performed essentially as described previously (HXX et al., 2024,Nucleic Acids Research) with the following modifications. After three rounds of selection, the enriched DNA library was amplified by fixed primers, and the recovered fragments were subjected to high-throughput sequencing. In the control reactions, library were incubated with protein storage buffer only.
SUB16280503
asdbd1_rep1