Comparative expression of soluble, active human kinases in specialized bacterial strains.

Abstract

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.