High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase
Year: 2026
Type: preprint
Abstract: Human Ca 2+ /calmodulin (CaM)-dependent protein kinase II alpha (CaMKIIα) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKIIα in bacterial cells. CaMKIIα was co-expressed with λ-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKIIα. Recombinant CaMKIIα was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKIIα has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKIIα, which could be suitable for subsequent biochemical and structural studies. Highlights Human CaMKIIα was successfully expressed in E. coli using autoinduction. Co-expression with λ-phosphatase maintained CaMKIIα predominantly in a non-phosphorylated state. The purified CaMKIIα showed no detectable Thr286 phosphorylation and retained Ca 2+ /calmodulin-dependent kinase activity.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7220471946