High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase
- 1BRIC-National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind Road, Pune 411007 Maharashtra, India
Abstract
Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) 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 CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-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 CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII 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 CaMKII, which could be suitable for subsequent biochemical and structural studies.
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BibTeX
@misc{yadav2026highyield,
title = {{High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase}},
author = {S. Yadav and L. D. Gaude and S. Kumar},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.02.756153},
url = {https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase
AU - S. Yadav
AU - L. D. Gaude
AU - S. Kumar
PY - 2026
DA - 2026/10/02
PB - bioRxiv
DO - 10.64898/2026.10.02.756153
UR - https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1
AB - Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) 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 CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-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 CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII 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 CaMKII, which could be suitable for subsequent biochemical and structural studies.
ER -