Production, purification, and crystallizationstrategies for recombinant EGFR and HER2 tyrosinekinase domains as a platform for structure-baseddrug discovery

The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinase domains (TKDs) are critical regulators of cell signaling and play central roles in cancer progression. Due to their therapeutic relevance, structural studies of EGFR-TKD and HER2-TKD are essential for structure-based drug discovery. In this thesis, recombinant EGFR-TKD and HER2-TKD were produced, purified, and subjected to initial crystallization trials. Both genes were cloned into the pET28a(+) expression vector and expressed in Escherichia coli (E. coli). Initial expression predominantly resulted in inclusion bodies; however, solubilization was successfully achieved using sarcosyl. Expression conditions, including temperature, inducer concentration, and induction time, were systematically optimized to improve yield and solubility. Purification strategies involved size-exclusion chromatography and a reverse affinity step to remove the SUMO tag, resulting in protein preparations suitable for crystallization. Following optimization, soluble forms of both EGFR-TKD and HER2-TKD were obtained. Initial crystallization experiments yielded promising crystals for EGFR-TKD, while optimization of crystal quality for HER2-TKD is ongoing. These results establish robust production and purification workflows, providing structural platforms that can be used for future drug screening efforts and the development of novel inhibitors targeting EGFR and HER2.