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

Rehman, Asma, Jarrott, Russell J., Whitten, Andrew E., King, Gordon J., Hu, Shu-Hong, Christie, Michelle P., Collins, Brett M. and Martin, Jennifer L. (2013) Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures. PloS One, 8 12: e83499.1-e83499.12. doi:10.1371/journal.pone.0083499


Author Rehman, Asma
Jarrott, Russell J.
Whitten, Andrew E.
King, Gordon J.
Hu, Shu-Hong
Christie, Michelle P.
Collins, Brett M.
Martin, Jennifer L.
Title Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures
Formatted title
Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures
Journal name PloS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2013-12-31
Year available 2013
Sub-type Article (original research)
DOI 10.1371/journal.pone.0083499
Open Access Status DOI
Volume 8
Issue 12
Start page e83499.1
End page e83499.12
Total pages 12
Editor Rizwan H. Khan
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Language eng
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.
Formatted 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.
Keyword Multidisciplinary Sciences
Science & Technology - Other Topics
MULTIDISCIPLINARY SCIENCES
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 535921
LE110100186
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
School of Chemistry and Molecular Biosciences
Institute for Molecular Bioscience - Publications
 
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