Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

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dc.contributor.authorVolpert, M.
dc.contributor.authorMangum, J.
dc.contributor.authorJamsai, D.
dc.contributor.authorD'Sylva, R.
dc.contributor.authorO'Bryan, M.
dc.contributor.authorMcIntyre, P.
dc.date.issued2014
dc.description.abstractWhile the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.
dc.description.statementofresponsibilityMarianna Volpert, Jonathan E. Mangum, Duangporn Jamsai, Rebecca D’Sylva, Moira K. O’Bryan & Peter McIntyre
dc.identifier.citationScientific Reports, 2014; 4(1):4217-1-4217-7
dc.identifier.doi10.1038/srep04217
dc.identifier.issn2045-2322
dc.identifier.issn2045-2322
dc.identifier.orcidO'Bryan, M. [0000-0001-7298-4940]
dc.identifier.urihttp://hdl.handle.net/2440/97416
dc.language.isoen
dc.publisherNature Publishing Group
dc.relation.granthttp://purl.org/au-research/grants/nhmrc/606562
dc.rightsThis work is licensed under a Creative Commons Attribution- NonCommercial-ShareAlike 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0
dc.source.urihttps://doi.org/10.1038/srep04217
dc.subjectCell Line
dc.titleEukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse
dc.typeJournal article
pubs.publication-statusPublished

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