title: Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse.
abstract: While 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.
authors.affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Marianna
authors.initials: M
authors.lastname: Volpert
authors.affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Mangum
authors.affiliation: Dept of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia.
authors.forename: Duangporn
authors.initials: D
authors.lastname: Jamsai
authors.affiliation: Dept of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia.
authors.forename: Rebecca
authors.initials: R
authors.lastname: D'Sylva
authors.affiliation: Dept of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia.
authors.forename: Moira K
authors.initials: MK
authors.lastname: O'Bryan
authors.affiliation: Health Innovations Research Institute, RMIT University, Bundoora, VIC, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: McIntyre
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: C489926:CRISP3 protein, human; C489927:Crisp3 protein, mouse; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D029589:Seminal Plasma Proteins