Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse
Files
(Published version)
Date
2014
Authors
Volpert, M.
Mangum, J.
Jamsai, D.
D'Sylva, R.
O'Bryan, M.
McIntyre, P.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
Scientific Reports, 2014; 4(1):4217-1-4217-7
Statement of Responsibility
Marianna Volpert, Jonathan E. Mangum, Duangporn Jamsai, Rebecca D’Sylva, Moira K. O’Bryan & Peter McIntyre
Conference Name
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.
School/Discipline
Dissertation Note
Provenance
Description
Access Status
Rights
This 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