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  • Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin

    Author
    Hu, Shu-Hong
    Gee, Christine L.
    Latham, C.F.
    Rowlinson, Scott W.
    Rova, Ulrika
    Jones, Alun
    Halliday, Judy A.
    Bryant, Nia J.
    James, David E.
    Martin, Jennifer L.
    Year published
    2003
    Metadata
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    Abstract
    Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin ...
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    Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c—both tagged and detagged—from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.
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    Journal Title
    Protein Expression and Purification
    Volume
    31
    Issue
    2
    DOI
    https://doi.org/10.1016/S1046-5928(03)00197-9
    Subject
    Biochemistry and Cell Biology not elsewhere classified
    Publication URI
    http://hdl.handle.net/10072/347924
    Collection
    • Journal articles

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