A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.
4 affiliations
- 1Department of Pharmacology, University of Cambridge, Cambridge, UK.
- 2Living Systems Institute, University of Exeter, Exeter, UK.
- 3Department of Biosciences, University of Exeter, Exeter, UK.
- 4Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
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.
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BibTeX
@article{bosetto2026generalizable,
title = {{A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.}},
author = {Federico Bosetto and Maria Zacharopoulou and Mark Bycroft and Monika Kish and Giovanna Zinzalla and Pamela J E Rowling and Stephen H McLaughlin and Jonathan J Phillips and Laura S Itzhaki and Ioanna Mela},
journal = {Protein science : a publication of the Protein Society},
year = {2026},
month = oct,
doi = {10.1002/pro.70825},
url = {https://europepmc.org/article/MED/42876396},
note = {CC BY},
}
RIS
TY - JOUR
TI - A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.
AU - Federico Bosetto
AU - Maria Zacharopoulou
AU - Mark Bycroft
AU - Monika Kish
AU - Giovanna Zinzalla
AU - Pamela J E Rowling
AU - Stephen H McLaughlin
AU - Jonathan J Phillips
AU - Laura S Itzhaki
AU - Ioanna Mela
PY - 2026
DA - 2026/10/30
JO - Protein science : a publication of the Protein Society
DO - 10.1002/pro.70825
UR - https://europepmc.org/article/MED/42876396
AB - 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.
SN - 0961-8368
SN - 1469-896X
ER -