Authors
Bosetto, F., Zacharopoulou, M., Bycroft, M., Kish, M., Zinzalla, G., Rowling, P., McLaughlin, S. H., Phillips, J. J., Itzhaki, L. S., Mela, I.
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 characterisation 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 E. 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 solubilisation tags. A GST His dual affinity strategy enabled efficient purification of the construct, whereas His 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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bioRxiv
The authors list and abstract were imported from bioRxiv on 13 Aug 2026.
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