{
  "abstract": "Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterization of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in Escherichia coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilization tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His6-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Pharmacology, University of Cambridge, Cambridge, UK.",
    "Living Systems Institute, University of Exeter, Exeter, UK.",
    "Department of Biosciences, University of Exeter, Exeter, UK.",
    "Medical Research Council Laboratory of Molecular Biology, Cambridge, UK."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Federico Bosetto",
      "orcid": "0000-0002-8217-2724"
    },
    {
      "affs": [
        0
      ],
      "name": "Maria Zacharopoulou",
      "orcid": "0000-0002-3660-2797"
    },
    {
      "affs": [
        0
      ],
      "name": "Mark Bycroft"
    },
    {
      "affs": [
        1,
        2
      ],
      "name": "Monika Kish"
    },
    {
      "affs": [
        0
      ],
      "name": "Giovanna Zinzalla",
      "orcid": "0000-0002-1130-4865"
    },
    {
      "affs": [
        0
      ],
      "name": "Pamela J E Rowling"
    },
    {
      "affs": [
        3
      ],
      "name": "Stephen H McLaughlin",
      "orcid": "0000-0001-9135-6253"
    },
    {
      "affs": [
        1,
        2
      ],
      "name": "Jonathan J Phillips"
    },
    {
      "affs": [
        0
      ],
      "name": "Laura S Itzhaki"
    },
    {
      "affs": [
        0
      ],
      "name": "Ioanna Mela",
      "orcid": "0000-0002-2914-9971"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/4yetyw3yx1p1f.bib",
  "cited_by": 0,
  "doi": "10.1002/pro.70825",
  "fulltext_url": "https://europepmc.org/articles/PMC13670019",
  "id": "4yetyw3yx1p1f",
  "issns": [
    "0961-8368",
    "1469-896X"
  ],
  "landing_url": "https://europepmc.org/article/MED/42876396",
  "language": "eng",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Protein purification and stability"
    ]
  ],
  "openalex": "W7222227590",
  "pdf_url": "https://europepmc.org/articles/PMC13670019?pdf=render",
  "pmcid": "PMC13670019",
  "pmid": "42876396",
  "published": "2026-10-30",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/4yetyw3yx1p1f.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:18:46Z",
      "native_id": "MED:42876396",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42876396"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Protein purification and stability",
      "subfield": "molecular-biology"
    }
  ],
  "title": "A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.",
  "title_text": "A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:18:46Z",
  "url": "https://research.draconitic.com/paper/4yetyw3yx1p1f",
  "venue": "Protein science : a publication of the Protein Society",
  "versions": []
}