title: Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.
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.
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Asma
authors.initials: A
authors.lastname: Rehman
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Jarrott
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Whitten
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Gordon J
authors.initials: GJ
authors.lastname: King
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Shu-Hong
authors.initials: SH
authors.lastname: Hu
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Michelle P
authors.initials: MP
authors.lastname: Christie
authors.affiliation: Division of Molecular Cell Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Brett M
authors.initials: BM
authors.lastname: Collins
authors.affiliation: Division of Chemistry and Structural Biology, University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Martin
mesh_terms: D000818:Animals; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D051938:Munc18 Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013816:Thermodynamics
chemical_list: D003062:Codon; D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C497943:Stxbp3 protein, mouse