{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "BRIC-National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Ganeshkhind Road, Pune 411007 Maharashtra, India"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "name": "S. Yadav"
    },
    {
      "name": "L. D. Gaude"
    },
    {
      "affs": [
        0
      ],
      "name": "S. Kumar"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/amdpwjkreb09v.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.02.756153",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1.full",
  "id": "amdpwjkreb09v",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "1312|Protein purification and stability"
    ]
  ],
  "openalex": "W7220471946",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-02",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/amdpwjkreb09v.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:26:02Z",
      "native_id": "10.64898/2026.10.02.756153",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.02.756153v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Protein purification and stability",
      "subfield": "molecular-biology"
    }
  ],
  "title": "High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase",
  "title_text": "High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase",
  "type": "preprint",
  "updated_at": "2026-10-30T12:26:02Z",
  "url": "https://research.draconitic.com/paper/amdpwjkreb09v",
  "venue": "bioRxiv",
  "versions": []
}