Production, purification, and crystallization of the recombinant HER2 tyrosine kinase domain (HER2-TKD)

Abstract

The human epidermal growth factor receptor 2 tyrosine kinase domain (HER2-TKD) is an important therapeutic target in oncology. In this study, we developed a practical and cost-effective approach for producing soluble recombinant HER2-TKD in Escherichia coli as a basis for structure-based drug discovery. The gene encoding HER2-TKD was cloned into a pET28a(+) expression vector and expressed in E. coli. Since the protein was initially obtained as inclusion bodies, solubilization with 1.5% sarcosyl was applied to recover it in soluble form. The purified protein was obtained through size-exclusion chromatography, followed by tag removal using reverse affinity purification. Crystallization trials were performed under more than 3000 conditions with commercial screening kits. Although most of the early crystals turned out to be salts rather than protein crystals, these observations underline the inherent challenges of HER2-TKD crystallization and highlight areas for further optimization. Overall, we establish a reproducible expression and purification workflow for HER2-TKD and provide a foundation for future cocrystallization experiments with small-molecule inhibitors and applications in structure-based drug screening.