Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid‐based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non‐membrane‐bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self‐associate upon mixing. Two fusion protein species (P3‐EGFR and P4‐EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5‐fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand‐induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.

Kinases act as molecular switches for cellular functions and are involved in multiple human pathogeneses, most notably cancer. There is a continuous need for soluble and active kinases for in-vitro drug discovery and structural biology purposes. Kinases remain challenging to express using Escherichia coli, the most widely utilized host for heterologous expression. In this work, four bacterial strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta, and Arctic Express, were chosen for parallel expression trials along with BL21 (DE3) complemented with folding chaperones DnaJ/K and GroEL/ES to compare their performance in producing soluble and active human kinases. Three representative diverse kinases were studied, Epidermal Growth Factor Receptor kinase domain, Aurora Kinase A kinase domain, and Mitogen-activated protein Kinase Kinase. The genes encoding the kinases were subcloned into pET15b bacterial plasmid and transformed into the bacterial strains. Soluble kinase expression was tested using different IPTG concentrations (1–0.05 mM) at varying temperatures (37°C– 10°C) and induction times (3–24 hours). The optimum conditions for each kinase in all strains were then used for 1L large scale cultures from which each kinase was purified to compare yield, purity, oligomerization status, and activity. Although using specialized strains achieved improvements in yield and/or activity for the three kinases, none of the tested strains was universally superior, highlighting the individuality in kinase expression.