TOPALAN, E.; Ciftci, H.; Demirci, H.

Background and Aim: The Epidermal Growth Factor Receptor Tyrosine Kinase Domain (EGFR-TKD) plays a key role in regulating cell growth and is closely linked to cancer progression. In this study, we set out to produce, purify, and crystallize recombinant EGFR-TKD in order to create a solid foundation for future drug screening and structure-based research. Materials and Methods: We used pET28a(+) plasmid and expressed EGFR-TKD in Escherichia coli. At first, most of the protein ended up in inclusion bodies, but with the help of sarcosyl, we were able to solubilize it effectively. To improve the amount and quality of the protein, we optimized several expression conditions, including temperature, inducer concentration, and induction time. The purification process included size-exclusion chromatography and a reverse affinity step to remove the SUMO tag, resulting in protein suitable for crystallization. Results: Following successful optimization, soluble EGFR-TKD was obtained, and initial crystals were successfully grown under defined conditions. These crystals represent a promising starting point for drug screening applications, providing a structural platform that can be further refined to support the discovery and development of novel EGFR inhibitors. Conclusion: This study presents an effective strategy for the production, purification, and initial crystallization of recombinant EGFR-TKD. The established workflow lays a solid foundation for future drug screening efforts and structural investigations targeting EGFR.