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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\u2010terminal plug and the Secretin\/TonB short N\u2010terminal domain. Each domain was expressed individually in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    and purified to homogeneity, adopting well\u2010folded 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\u2010His dual\u2010affinity strategy enabled efficient purification of the construct, whereas His6\u2010tag alone resulted in insoluble protein and HLT\u2010tag fusions suffered from non\u2010specific proteolysis. Biophysical analyses revealed that the Secretin\/TonB short N\u2010terminal domain remains stably folded within the fusion construct, while the N\u2010terminal 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\u2010screening applications, including aptamer and nanobody discovery.\n                  <\/jats:p>","DOI":"10.1002\/pro.70825","type":"journal-article","created":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T08:22:21Z","timestamp":1792052541000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A generalizable method for the purification and biophysical characterization of bacterial membrane receptors"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8217-2724","authenticated-orcid":false,"given":"Federico","family":"Bosetto","sequence":"first","affiliation":[{"name":"Department of Pharmacology University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3660-2797","authenticated-orcid":false,"given":"Maria","family":"Zacharopoulou","sequence":"additional","affiliation":[{"name":"Department of Pharmacology University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Bycroft","sequence":"additional","affiliation":[{"name":"Department of Pharmacology University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Kish","sequence":"additional","affiliation":[{"name":"Living Systems Institute University of Exeter  Exeter UK"},{"name":"Department of Biosciences University of Exeter  Exeter UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1130-4865","authenticated-orcid":false,"given":"Giovanna","family":"Zinzalla","sequence":"additional","affiliation":[{"name":"Department of Pharmacology University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pamela J. 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