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.

The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly

“…The importance of the Sx4 N-peptide for binding to Munc18c was confirmed once again using ITC, where binding was detected for HMunc18c (Munc18c with an N-terminal His 6 -tag) and Sx4 1-275 -T4L-His, but not for HMunc18c and Sx4 30-275 -T4L-His ( S1 Table ). The ITC determined binding affinity, K d , of 100 nM for the HMunc18c:Sx4 1-275 -T4L-His interaction ( S1 Table , S1 Fig ) is similar to that reported previously for the interaction between Munc18c and Sx4 1-275 -His ( K d 95–104 nM) [ 17 , 39 ]. These results indicate that the T4L fusion does not affect the binding affinity of Sx4 for Munc18c.…”

“…Mouse HMunc18c (N-terminal His 6 -tag), HTMunc18c (N-terminal His 6 -tag, TEV cleavage site, referred to as detagged Munc18c after the His tag is removed) and HL-Munc18c (N-terminal His 6 -tag, with a 53 amino acid linker) constructs were also prepared as described by Rehman et al . [ 39 ]. The 53 amino acid linker has the following sequence: .…”

“…Munc18c proteins were expressed in E . coli and purified as described previously [ 39 ], with the following changes. Tris was replaced with 50 mM phosphate (pH 8.0) in all purification buffers.…”

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins

“…Mouse Munc18c (residues 1–592) with a non-cleavable N-terminal 6x-His tag was generated using the pQE30 vector. This was modified to contain a TEV protease cleavage site to enable removal of the 6x-His tag, [ 42 ] and expressed in E . coli .…”

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

“…Using a baculovirus expression system rather than a bacterial expression system can potentially introduce modifications to the protein, for example, phosphorylation or glycosylation. Recently we observed that bacterially expressed Munc18c interacts with Sx4 (residues 1-275) with similar affinity to baculovirus-expressed Munc18c, showing that the Munc18c/ Sx4 interaction is not dependent on such modifications if they are present (Rehman et al, 2013). However, whether such modifications are important for Munc18 function in promoting or inhibiting fusion is not well studied and needs to be considered in the future.…”