BPS2026 – Recombinant expression and purification of NF-κB p50/cRel heterodimers for DNA-binding studies

The NF-κB family of transcription factors regulates genes involved in immunity, inflammation, and other biological processes. Members of the NF-κB family can form homo- or heterodimers, which contribute to specific responses to various stimuli. Three NF-κB subunits, RelA, RelB, and c-Rel, contain long intrinsically disordered regions (IDRs) that function as transcription activation domains. The p50/RelA heterodimer has been previously studied in its full-length form, but full-length p50/RelB and p50/c-Rel remain uncharacterized biophysically. This project aims to do a comparison study between the full length p50/RelA, p50/RelB, and p50/c-Rel IDRs. We optimized the protocol to express recombinant p50/c-Rel in E. coli and purify the proteins using Ni 2+ affinity, ion exchange, and size exclusion chromatography. SDS-PAGE analysis confirmed the successful isolation of both protein subunits at the expected molecular weights. Using this protocol, we will produce full-length p50/c-Rel to be used in DNA- and co-activator-binding studies. These findings will establish the groundwork to compare the c-Rel IDR with IDRs from other NF-κB heterodimers, providing insights into the roles of these IDRs in NF-κB signaling specificity and immune regulation.