Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

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 structu...

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Autores principales:	Asma Rehman, Russell J Jarrott, Andrew E Whitten, Gordon J King, Shu-Hong Hu, Michelle P Christie, Brett M Collins, Jennifer L Martin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1f122e2f27a84a0b9f9201719cfb7460
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spelling	oai:doaj.org-article:1f122e2f27a84a0b9f9201719cfb74602021-11-18T08:39:36ZMilligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.1932-620310.1371/journal.pone.0083499https://doaj.org/article/1f122e2f27a84a0b9f9201719cfb74602013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24391775/?tool=EBIhttps://doaj.org/toc/1932-6203Vesicle 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.Asma RehmanRussell J JarrottAndrew E WhittenGordon J KingShu-Hong HuMichelle P ChristieBrett M CollinsJennifer L MartinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e83499 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Asma Rehman Russell J Jarrott Andrew E Whitten Gordon J King Shu-Hong Hu Michelle P Christie Brett M Collins Jennifer L Martin Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
description	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.
format	article
author	Asma Rehman Russell J Jarrott Andrew E Whitten Gordon J King Shu-Hong Hu Michelle P Christie Brett M Collins Jennifer L Martin
author_facet	Asma Rehman Russell J Jarrott Andrew E Whitten Gordon J King Shu-Hong Hu Michelle P Christie Brett M Collins Jennifer L Martin
author_sort	Asma Rehman
title	Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
title_short	Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
title_full	Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
title_fullStr	Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
title_full_unstemmed	Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
title_sort	milligram quantities of homogeneous recombinant full-length mouse munc18c from escherichia coli cultures.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/1f122e2f27a84a0b9f9201719cfb7460
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Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

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