Production, purification, and ınitial crystallization of recombinant epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) as a structural basis for future drug screening studies

Abstract

The epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) is a key regulator of intracellular signaling events that control cell proliferation. Aberrant EGFR activation is closely associated with the development of non-small cell lung cancer (NSCLC). In this study, recombinant EGFR-TKD was expressed in Escherichia coli Rosetta™ 2 (DE3) cells using a pET28a(+) expression plasmid carrying an N-terminal 6×His-SUMO- tag. During expression, a large proportion of the protein accumulated in inclusion bodies; therefore, we employed a solubilization approach. Treatment with 1.5% sarcosyl reproducibly yielded a soluble protein fraction compatible with subsequent purification steps. Optimizing induction parameters, including temperature, IPTG concentration, and expression duration, improved both the yield and solubility of the protein. Following size-exclusion chromatography, the monomeric protein fraction was isolated and used for crystallization trials, which resulted in the formation of small but well-defined microcrystals under several conditions. Although these crystals did not yet provide diffraction suitable for structure determination, their reproducible appearance indicates that the obtained EGFR-TKD is structurally competent for crystallization trials. Overall, the workflow establishes a practical and reproducible bacterial expression and purification strategy that forms a basis for continued crystallization optimization and structure-based inhibitor development targeting EGFR-driven cancers.