Overcoming Discovery Challenges for Highly Disordered Transcription Factors Using eProtein Discovery™

Previously using cell based techniques, the expression and purification of TF L, especially in its full-length form or specific functional domains, have proven challenging due to the protein’s complex structure. A notoriously difficult to express protein with DNA binding (homeodomain) and LIM Domains and intrinsically disordered regions. These challenges have severely restricted the ability to conduct detailed structural and functional studies of TF L, thus impeding the development of targeted therapies. TF L is particularly crucial in developmental processes such as neural differentiation, hematopoiesis, and organogenesis. This Application Note, covers how we employed the eProtein Discovery System to successfully express and purify full-length Transcription Factor L (TF L), along with a domain of unknown function (L2) and its binding domain (L3).

From reading this application note, you’ll learn:

• By utilizing solubility tags and Cell-free Blends enriched with Zn2+ and disulfide bond-forming agents, we optimized the production of soluble TF L protein.
• The DNA-binding functionality of the purified protein was evaluated using 488 Atto dye-labeled double-stranded DNA containing the TF L binding site.
• Our results not only address the longstanding challenges associated with TF L synthesis but also open new avenues for therapeutic development, positioning the eProtein Discovery System as a transformative tool in the study and production of complex transcription factors.