dc.contributor.author | Rehman, Asma | |
dc.contributor.author | Jarrott, Russell J | |
dc.contributor.author | Whitten, Andrew E | |
dc.contributor.author | King, Gordon J | |
dc.contributor.author | Hu, Shu-Hong | |
dc.contributor.author | Christie, Michelle P | |
dc.contributor.author | Collins, Brett M | |
dc.contributor.author | Martin, Jennifer L | |
dc.date.accessioned | 2018-01-19T05:12:51Z | |
dc.date.available | 2018-01-19T05:12:51Z | |
dc.date.issued | 2013 | |
dc.identifier.issn | 1932-6203 | |
dc.identifier.doi | 10.1371/journal.pone.0083499 | |
dc.identifier.uri | http://hdl.handle.net/10072/171862 | |
dc.description.abstract | Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c
is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose
tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to
maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we
investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports
describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations.
Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature
expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch
virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–
2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture.
The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In
summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with
previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies. | |
dc.description.peerreviewed | Yes | |
dc.language | English | |
dc.language.iso | eng | |
dc.publisher | Public Library of Sciences | |
dc.relation.ispartofpagefrom | e83499-1 | |
dc.relation.ispartofpageto | e83499-12 | |
dc.relation.ispartofissue | 12 | |
dc.relation.ispartofjournal | PLoS One | |
dc.relation.ispartofvolume | 8 | |
dc.subject.fieldofresearch | Biochemistry and cell biology not elsewhere classified | |
dc.subject.fieldofresearchcode | 310199 | |
dc.title | Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures | |
dc.type | Journal article | |
dc.type.description | C1 - Articles | |
dc.type.code | C - Journal Articles | |
dcterms.license | https://creativecommons.org/licenses/by/4.0/ | |
dc.description.version | Version of Record (VoR) | |
gro.rights.copyright | © 2013 Rehman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. | |
gro.hasfulltext | Full Text | |
gro.griffith.author | Martin, Jennifer | |