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Researchers developed a generalizable method for expressing and purifying soluble domains from HasR, a bacterial membrane receptor crucial for iron acquisition in Pseudomonas aeruginosa. They successfully produced two isolated HasR domains and a dual-domain fusion construct using a GST-His dual affinity tag strategy, with biophysical characterization revealing that the fusion construct maintains partial structural integrity. The study provides a systematic workflow for obtaining soluble membrane protein domains suitable for structural studies and ligand discovery applications.
Why it matters
This work addresses a major technical challenge in studying bacterial membrane proteins, which are difficult to work with but represent important drug targets. The validated constructs and purification strategy can facilitate the development of new therapeutics targeting P. aeruginosa infections, particularly relevant given rising antibiotic resistance.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
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.