Expression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptor

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dc.contributor.authorMcRae, MSen_AU
dc.contributor.authorWang, Ben_AU
dc.contributor.authorHyson, RGen_AU
dc.contributor.authorSiddiquee, Ren_AU
dc.contributor.authorDuff, APen_AU
dc.contributor.authorAtaide, SFen_AU
dc.contributor.authorKwan, AHen_AU
dc.date.accessioned2025-01-10T04:02:42Zen_AU
dc.date.available2025-01-10T04:02:42Zen_AU
dc.date.issued2022-10en_AU
dc.date.statistics2024-11-14en_AU
dc.description.abstractThe Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies. © 2022 Elsevier Inc. All rights reserved.en_AU
dc.description.sponsorshipWe acknowledge Yupaporn Phonok for collecting the ESI-MS spectra and Dr Bill Bubb for proofreading the manuscript. We acknowledge access to real-time PCR, NMR and MS instruments at Sydney Analytical Core Research Facility and the Chemistry Mass Spectrometry Unit at the University of Sydney and thank the Facility staff scientists Biswaranjan Mohanty and Nicholas Proschogo for access and assistance. Band excision from an SDS-PAGE gel, trypsin digestion and MALDI-TOF was performed by Sydney Mass Spectrometry Core Research Facility. 2H15N-SRα-NG was produced under proposal NDF8822 at the National Deuteration Facility, which is partly supported by the National Collaborative Research Infrastructure Strategy ― an initiative of the Australian Government.en_AU
dc.format.mediumPrint-Electronicen_AU
dc.identifier.articlenumber106121en_AU
dc.identifier.citationMcRae, M. S., Wang, B., Hyson, R. G., Siddiquee, R., Duff, A. P., Ataide, S. F., & Kwan, A. H. (2022). Expression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptor. Protein Expression and Purification, 198, 106121. doi:10.1016/j.pep.2022.106121en_AU
dc.identifier.issn1046-5928en_AU
dc.identifier.issn1096-0279en_AU
dc.identifier.journaltitleProtein Expression and Purificationen_AU
dc.identifier.urihttps://doi.org/10.1016/j.pep.2022.106121en_AU
dc.identifier.urihttps://apo.ansto.gov.au/handle/10238/15899en_AU
dc.identifier.volume198en_AU
dc.languageEnglishen_AU
dc.language.isoenen_AU
dc.publisherElsevieren_AU
dc.subjectPurificationen_AU
dc.subjectReceptorsen_AU
dc.subjectStrontiumen_AU
dc.subjectNuclear magnetic resonanceen_AU
dc.subjectPlasmaen_AU
dc.subjectProteinsen_AU
dc.subjectDrugsen_AU
dc.subjectScreeningen_AU
dc.titleExpression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptoren_AU
dc.typeJournal Articleen_AU
dcterms.dateAccepted2022-05-26en_AU
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